WO2012092295A2 - Diagnostic optique de cellules anormales - Google Patents

Diagnostic optique de cellules anormales Download PDF

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Publication number
WO2012092295A2
WO2012092295A2 PCT/US2011/067425 US2011067425W WO2012092295A2 WO 2012092295 A2 WO2012092295 A2 WO 2012092295A2 US 2011067425 W US2011067425 W US 2011067425W WO 2012092295 A2 WO2012092295 A2 WO 2012092295A2
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WO
WIPO (PCT)
Prior art keywords
sample
substrate
light
layer
high intensity
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Application number
PCT/US2011/067425
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English (en)
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WO2012092295A3 (fr
Inventor
Maria NAVAS-MORENO
Zeev Valentine Vardeny
Josef T. Prchal
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The University Of Utah Research Foundation
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Publication of WO2012092295A2 publication Critical patent/WO2012092295A2/fr
Publication of WO2012092295A3 publication Critical patent/WO2012092295A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present application is directed generally toward optical observation of cellular abnormalities. More specifically, embodiments are directed towards using spectroscopic analysis to detect abnormal cells.
  • MPDs Myeloproliferative diseases
  • PV polycythemia vera
  • ET essential thrombocythemia
  • myelofibrosis Characteristics of these diseases are bone marrow hypercellularity, predisposition to thrombosis and hemorrhage, and an increased risk of evolution towards universally fatal acute leukemia.
  • MPDs The diagnosis of MPDs is quite challenging. PV is often difficult and expensive to differentiate from reactive polycythemic, nonmalignant disorders; whereas, ET is largely diagnosed by exclusion of other conditions with increased platelet count due to the lack of specific tests.
  • the three MPDs discussed above often share a somatic clonal mutation of a tyrosine kinase gene that encodes for JAK2. Unfortunately, whereas this mutation is diagnostic for the presence of a myeloproliferative disorder but not its type, its absence also does not exclude the existence of MPD. The phenotype of MPDs is known. However, genetic and post- genetic abnormalities, and host modifiers that could be detected, are yet to be discovered.
  • the JAK2 mutation is present in about 90% of the Polycythemia Vera patients, but only present in about 50% of the Essential Thrombocythemia patients and 50% of the Idiopatic Myelofibrosis patients.
  • a patient may have an MPD without exhibiting the mutation.
  • the methods are also further complicated by the fact that the JAK2 mutation is also found in other disorders.
  • the present application provides systems and methods for assisting in the diagnosis of various conditions by utilizing Raman scattering, or surface-enhanced Raman scattering, spectroscopy and auto-florescence signals scattered and emitted from a sample.
  • the samples are often tissue, blood or other bodily fluids from a subject, such as a human patient, in which an abnormal sample may be used as indicative of that patient having an MPD.
  • the received signals are then compared to control values in order to recognize differences between a normal sample and the abnormal sample.
  • measurements are made on a single cell level and samples are deposited on substrate having a layer of reflective nanostructures disposed thereon.
  • An embodiment of the invention may be described as a system including a coherent light source configured to emit high intensity light, and a sample substrate oriented to be impinged by the high intensity light from the coherent light source.
  • the system includes an optical element which is configured to direct light from the light source to the substrate, and subsequently direct the scattered and emitted light from the sample to a spectrometer, which may be configured to receive a light signal corresponding to the interaction of the high intensity light with the sample and substrate, and to output data signals corresponding to the spectrum of the scattered and emitted light from the sample.
  • the system may include a processing device configured to receive the data signals corresponding to the spectrum of scattered and emitted light, wherein the processing device is configured to compare spectral results of a tissue sample disposed on the sample substrate with a control data set and generate a probability value that an abnormality exists within a cell of the tissue sample.
  • a substrate contains a base substrate layer.
  • An example of the base layer may be glass, such as a borosilicate.
  • the substrate further includes a layer of reflective particles disposed on the base substrate layer.
  • preparation of the substrate is undertaken prior to allowing a tissue sample to contact the substrate. This preparation may include removing substantially all carbon-based species from the substrate by exposing the sample substrate to ultraviolet (UV) radiation and exposing the sample substrate to an oxygen air flow. In some cases, the exposure of UV radiation and air flow is implemented contemporaneously.
  • UV ultraviolet
  • the above systems and methods are utilized to conduct hypothesis testing.
  • This testing may define a null hypothesis where samples from both patient groups and control groups are the same. A probability is then calculated, with the value representing whether the null hypothesis is correct. If the probability value is low, then the null hypothesis can be safely discarded.
  • a tissue sample may be determined to be affected by diseases, such as MPD.
  • FIGURE 1 illustrates a block diagram of a system in accordance with an embodiment of the present invention
  • FIGURE 2 illustrates an example implementation of a sample substrate in accordance with an embodiment of the present invention.
  • FIGURE 3 illustrates a flowchart for a method process in accordance with an embodiment of the present invention.
  • Figure 1 illustrates an embodiment of system 100 configured to implement the teachings described herein.
  • System 100 includes a laser 110 configured to emit coherent light 111 which is to be directed at sample substrate 120.
  • coherent light 11 1 is directed through an optical element 130.
  • Optical element 130 is configured to direct or focus coherent light 1 1 1 to sample substrate 120.
  • optical element 130 comprises a beam splitter 131 to accomplish the directing and focusing of coherent light to sample substrate 120.
  • optical element 130 may include a microscope portion which allows a user to view cells located on sample substrate 120.
  • Scattered and emitted light 112 is collected by optical element 130 where it is then directed to spectrometer 140.
  • scattered and emitted light 1 12 may be directed to spectrometer 140 utilizing beam splitter 131.
  • the scattered and emitted light 112 from optical element 130 to spectrometer 140 may be conveyed by various means, e.g., optical coupling, fiber optic cable, and the like.
  • Spectrometer 140 may function to measure the light spectrum of the scattered and emitted light 112 and to output data signal 113 to a computing system 150 for subsequent processing.
  • computing system 150 is configured only to process received signals. In other embodiments, computing system 150 is configured to control one or more stages of the acquisition of data. For example, computing system 150 may be utilized to control the timing and intensity of the light emitted from laser 1 10. Additionally, computing system 150 may be utilized to calibrate optical element 130, control the focus of coherent light 11 1 and scattered and emitted light 112, and the like. Further, computing system 150 may be utilized to control the area of the sample substrate which is exposed to coherent light 111. This control may be accomplished, for example, by moving substrate 120, portions of optical element 130, etc.
  • FIG. 2 illustrates an example implementation of a sample substrate 200 in accordance with an embodiment of the present invention.
  • Substrate 200 comprises a base substrate layer 201, and reflective particles 202.
  • Sample 203 may be deposited onto substrate 200 for subsequent analysis.
  • base substrate layer 201 is formed from a glass material, such as a high quality borosilicate. Such materials generally do not fluoresce when impinged with laser light, and generally do not have an adverse impact on tissue, blood or other bodily fluid samples, such as sample 203. It is noted that base substrate layer 201 may be made from any material which will have either no effect, or at least have a controllable effect, on the optic signals incident to and reflected from substrate 200. Additionally the materials of base substrate layer 201 will preferably not damage/contaminate, or will create minimal
  • Reflective particles 202 may be implemented with any material with optical properties that are suitable for carrying out the functionality described herein. Particles which reflect light with harmonic resonances in the visible light spectrum are preferable at the present time due to the current spectroscopic techniques and the ability to analyze light in the visible range. Examples of such reflective particles include elements such as gold, silver and copper. Reflective particles may be deposited onto base substrate layer 201 using any deposition techniques such as chemical reduction of a metal salt, electrochemical reduction, nanolithography and nanoimprint and the like.
  • base substrate layer 201 is glass
  • reflective particles 202 are embodied as deposited silver nanoparticles with glucose reduction of silver nitrate.
  • this example embodiment Prior to utilizing substrate 200, this example embodiment uses a technique to clean the silver nanoparticles in order to prevent the silver nanoparticles from being contaminated after they have been deposited on substrate layer 201.
  • one embodiment exposes substrate 200 to UV radiation and a flow of oxygen for a specified period of time (usually less than a minute) in order to take substantially all of the carbon species out of substrate 200. Because sample 203 will be from a carbon-based species, taking the carbon out of substrate 200 effectively cleans the substrate, including silver particles 202, to assure better light scattering enhancement performance. It is noted that when carbon contamination is present, it may be difficult to distinguish the sample and contaminants in the spectroscopic results.
  • the substrate cleaning is done in a clean room atmosphere.
  • embodiments may implement such a cleaning in a nitrogen atmosphere in order to assist in the prevention of carbon contamination.
  • cleaning procedures such as described above, may vary based on the type of materials used for base substrate layer 201 and/or reflective particles 202. For example, when cleaning gold
  • nanoparticles extended UV radiation and/or oxygen flow time, or different techniques all together (e.g. mechanical polishing followed by sonication and chemical polishing), may be utilized to minimize carbon contamination.
  • substrate 200 may be implemented with commercially available products, or specifically made in a manner which is optimized for a particular test sequence.
  • reflective particles 202 may be deposited in particular geometrical patterns. Spacing between particles, aspect ratio and density changes will all have an effect on the enhancement of signal and the type of subsequent processing which may be implemented.
  • some embodiments may tailor the distribution of reflective particles 202 based on the particular use of substrate 200 or sample 203 being analyzed.
  • Hot spots are helpful because regions of substrate 200 which include hot spots constitute regions with the strongest enhancement of scattered light, and therefore provide additional measurement data.
  • Sample 203 may be any sample that is capable of being analyzed according to the principles discussed herein.
  • sample 203 is a blood sample.
  • Such a sample may be separated by centrifugation and by density medium.
  • granulocytes are collected, which are a type of white blood cell.
  • Granulocytes are the daughter cells of the progenitor cell in which the mutation(s) are believed to be acquired.
  • the granulocytes are the cells that are affected by the mutation, and are, therefore, the cells that are being observed.
  • embodiments may be implemented with any type of cell such as monocytes, lymphocytes and platelets, or slices and/or isolated cells from cancerous and other diseased tissues, when attempting to diagnose other diseases.
  • the solution is smeared onto a substrate, such as substrate 200 and may then be air dried.
  • the dried sample is then ready to be placed into the spectrometer system, such as system 100.
  • the intensity of coherent light 11 1 has various effects on sample 203.
  • a system will use the minimum intensity that will produce the scattered light signal.
  • Minimum intensity generally corresponds to the intensity that will yield a scattered signal without carbonization of the sample.
  • high intensity exposure time of a sample is approximately one second.
  • the spectrometer system then receives the light emitted and scattered from sample 203 and provides the results of the scattering technique to a processing system, such as computer system 150. Once the processing system obtains the information about the sample, the information may then be compared to a control sample, or set of samples to determine if any abnormalities exist.
  • approximately 30 cells per subject are collected, whereas in other embodiments it may be preferable to increase this number.
  • the ratio of intensities of different parts of the spectrum between the sample cells and control cells are observed. Based on these ratios, embodiments may utilize hypothesis testing to determine whether the control group and patient group are different by observing one or more ratios in the spectrum for areas of the spectrum that show a significant difference in results.
  • control group which is able to provide tissue samples are tested and the resulting spectrum is observed.
  • the control group consists of greater than 20 people. It is appreciated that the number of people utilized in the control group will affect the statistical analysis when comparing samples to a control value.
  • a threshold value is then set, above (or below) which an individual may be considered a patient, and below (or above) which an individual may be considered a control value (e.g., having a normal sample).
  • the sensitivity and specificity of the techniques are integrated for different particular thresholds. With these values, an Receiver Operating Characteristic (ROC) curve is constructed to provide the threshold value.
  • the ROC shows the tradeoff between sensitivity and specificity for a diagnostic technique as well as the accuracy of the technique which is represented by the area under the curve. In general, areas larger than 0.8 are considered as good, and the area under the ROC curve for techniques described herein have been noted as being around 0.896.
  • a threshold value for comparison is set and may be utilized to compare results from various samples. Examples of an ROC curve are found in Appendix A.
  • Appendix A illustrates Glucose and Phospholipid plots.
  • solutions of glucose and phospholipids were drop cast onto the reflective nanoparticles and surfaced-enhanced Raman measurements were performed.
  • An attempt to reconstruct the cells spectra based on the spectra of the glucose and the phospholipids was shown successful for the patients but not for the controls. This result may indicate that differences between controls and patients may arise from various contents of glucose and/or phospholipids within the cells.
  • embodiments When comparing samples, embodiments have evaluated sensitivity (% of correctly diagnosed positives) and specificity (% of correctly identified negatives) and have shown to be as accurate as determining these values with as much as a 85% accuracy; this value may be increased in combination with another diagnosis technique as summarized in the above. Additionally, when hypothesis testing between different MPD disorders, it can be determined that ET may be present as opposed to PV or IMF by examining the intensity ratios in different areas of the spectrum.
  • the information obtained may function to eliminate or at least reduce the number of additional tests traditionally undertaken by a patient.
  • FIG. 3 illustrates a method 300 flowchart in accordance with an embodiment of the present invention.
  • a blood/tissue/bodily fluid sample is collected from a patient.
  • the sample is then separated at block 302, such as by centrifugation, into different cell types.
  • a particular cell type of interest is collected and placed into a solution at block 303, which is smeared onto a substrate.
  • One or all of the cells on the substrate is then exposed to a coherent light source at block 304.
  • Spectral information regarding the exposed cells is then collected and directed towards a spectrometer at block 305. This may be done iteratively at block 306 until information for a pre-determined number of cells is obtained. In some embodiments approximately 30 cells are exposed and information corresponding to these cells is collected.
  • Embodiments may then output the collected information to a processing device for further analysis at block 307.
  • the coherent light source is smaller than the cell itself. In such embodiments it may be preferable to aim the laser at the middle of the cell.
  • the scattered light information may be directed to the spectrometer using any means to convey a signal, such as optic coupling, transmission via fiber optic cable, and the like.
  • the spectrometer may be equipped with a charge-coupled device (CCD) which functions to send an electrical signal representative of the received light signal to a processing device.
  • CCD charge-coupled device
  • processing may be implemented as described above. It is further noted that the processing described above, and the overall functionality and control of the spectroscopy system (such as system 100) may be implemented manually, with the assistance of software configured to execute on a processing device, or as a function of both. The description herein is not intended to be limiting as to how such systems and methods may be implemented.

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  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)

Abstract

La présente invention se rapporte à une aide au diagnostic de diverses conditions en utilisant la diffusion Raman ou une spectroscopie par diffusion Raman exaltée de surface et des signaux d'auto-fluorescence dispersés et émis depuis un échantillon. Selon certains modes de réalisation, l'échantillon est un échantillon de sang ou de tissu provenant d'un patient humain, l'échantillon anormal pouvant être indicatif du fait que le patient souffre d'une maladie myéloproliférative (MPD, Myelo-Proliferative Disease). Les signaux lumineux sont comparés à des valeurs de contrôle afin de reconnaître les différences entre un échantillon normal et un échantillon anormal. De plus, selon certains modes de réalisation, des mesures sont réalisées au niveau monocellulaire et des échantillons sont déposés sur un substrat qui présente une couche de matériau réfléchissant disposée sur ce dernier.
PCT/US2011/067425 2010-12-30 2011-12-27 Diagnostic optique de cellules anormales WO2012092295A2 (fr)

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US201061428697P 2010-12-30 2010-12-30
US61/428,697 2010-12-30

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WO2012092295A3 WO2012092295A3 (fr) 2012-11-22

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103190917A (zh) * 2013-04-10 2013-07-10 重庆绿色智能技术研究院 一种基于激光拉曼技术的血糖仪
RU2546518C2 (ru) * 2013-05-23 2015-04-10 Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) Способ анализа мембраносвязанного гемоглобина в эритроцитах с помощью спектроскопии гигантского комбинационного рассеивания на наноструктурированных покрытиях
CN110057822A (zh) * 2019-04-23 2019-07-26 中国科学院深圳先进技术研究院 一种用于病理检查的光学成像系统

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5991653A (en) * 1995-03-14 1999-11-23 Board Of Regents, The University Of Texas System Near-infrared raman spectroscopy for in vitro and in vivo detection of cervical precancers
US6002476A (en) * 1998-04-22 1999-12-14 Chemicon Inc. Chemical imaging system
GB9907688D0 (en) * 1999-04-06 1999-05-26 Univ Belfast Solid matrices for surface-enhanced Raman spectroscopy
US7428045B2 (en) * 2002-01-10 2008-09-23 Chemimage Corporation Raman spectral analysis of pathogens
US7450227B2 (en) * 2004-09-22 2008-11-11 The Penn State Research Foundation Surface enhanced Raman spectroscopy (SERS) substrates exhibiting uniform high enhancement and stability
US20100129623A1 (en) * 2007-01-29 2010-05-27 Nanexa Ab Active Sensor Surface and a Method for Manufacture Thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103190917A (zh) * 2013-04-10 2013-07-10 重庆绿色智能技术研究院 一种基于激光拉曼技术的血糖仪
RU2546518C2 (ru) * 2013-05-23 2015-04-10 Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) Способ анализа мембраносвязанного гемоглобина в эритроцитах с помощью спектроскопии гигантского комбинационного рассеивания на наноструктурированных покрытиях
CN110057822A (zh) * 2019-04-23 2019-07-26 中国科学院深圳先进技术研究院 一种用于病理检查的光学成像系统
WO2020215802A1 (fr) * 2019-04-23 2020-10-29 中国科学院深圳先进技术研究院 Système d'imagerie optique pour examen pathologique

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