[go: up one dir, main page]

WO2012124763A1 - Procédé d'évaluation de tissu - Google Patents

Procédé d'évaluation de tissu Download PDF

Info

Publication number
WO2012124763A1
WO2012124763A1 PCT/JP2012/056689 JP2012056689W WO2012124763A1 WO 2012124763 A1 WO2012124763 A1 WO 2012124763A1 JP 2012056689 W JP2012056689 W JP 2012056689W WO 2012124763 A1 WO2012124763 A1 WO 2012124763A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluorescent
bright spots
staining
recognition site
nanoparticles
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2012/056689
Other languages
English (en)
Japanese (ja)
Inventor
岡田　尚大
秀樹 郷田
健作 高梨
拓司 相宮
中野　寧
幸祐 権田
元博 武田
憲明 大内
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tohoku University NUC
Konica Minolta Medical and Graphic Inc
Original Assignee
Tohoku University NUC
Konica Minolta Medical and Graphic Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tohoku University NUC, Konica Minolta Medical and Graphic Inc filed Critical Tohoku University NUC
Priority to JP2013504769A priority Critical patent/JP5863057B2/ja
Publication of WO2012124763A1 publication Critical patent/WO2012124763A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms

Definitions

  • the present invention relates to a tissue evaluation method of immunohistochemistry. More specifically, the present invention relates to a tissue evaluation method with higher accuracy by evaluation with a wide field of view.
  • the collected tissue is first dehydrated to fix it, blocked with paraffin, cut into thin slices with a thickness of 2 to 8 ⁇ m, the paraffin removed, stained, and observed under a microscope.
  • a pathologist makes a diagnosis based on morphological information such as changes in the size and shape of cell nuclei and changes in pattern as a tissue, and staining information.
  • image digitization technology in the field of pathological diagnosis, information necessary for pathologists to perform pathological diagnosis from pathological images input as digital color images using a microscope, digital camera, etc. Automated pathological diagnosis support devices that extract, measure and display have become widespread.
  • Patent Document 1 discloses a nucleus / cytoplasm distribution estimation unit that specifies a cell nucleus region and a cytoplasm region from a pathological image, and a glandular space extraction that specifies a glandular cavity region (a region that hardly includes cellular tissue) from a pathological image.
  • a pathological diagnosis support apparatus having the above is disclosed.
  • Patent Document 2 a pathological specimen is stained with two types of dyes that selectively stain a normal site and a cancer site, and the staining density is evaluated from a spectral image using Lambert-Beer's law.
  • a method for detecting cancer cells that determines the presence or absence of cancer cells is disclosed.
  • the tissue staining method is a conventional dye staining method (for example, hematoxylin-eosin staining) or a dye staining method using an enzyme (for example, DAB staining). It is greatly influenced by environmental conditions such as time, and does not make full use of the accurate quantitative measurement performance of the pathological diagnosis support apparatus.
  • Non-Patent Document 1 fluorescent dyes with high quantitative performance are used for tissue staining as a labeling reagent in place of the dye (see Non-Patent Document 1), but the emission luminance is darker than that of the autofluorescence emitted by the tissue. The biomarker cannot be automatically determined by the light emission level.
  • trastuzumab which is an antibody of HER2 (human epidermal growth factor receptor type 2)
  • HER2 human epidermal growth factor receptor type 2
  • HER2 was discovered as an oncogene having a similar structure to the human epidermal growth factor receptor (EGFR) gene.
  • the HER2 protein encoded by the HER2 gene is a receptor-type glycoprotein that penetrates the cell membrane, and is composed of three domains: extracellular, transmembrane, and intracellular. When a growth factor is bound, it is activated by phosphorylation of tyrosine residues, and is involved in cell growth and malignant transformation through a signal transduction pathway.
  • Overexpression of HER2 is also observed in lung cancer, colon cancer, stomach cancer, bladder cancer and the like. In breast cancer, 90% of the cause of HER2 protein overexpression is amplification of the HER2 gene.
  • transcription abnormalities and post-transcriptional protein synthesis abnormalities are also considered causes.
  • HER2 is considered to be a prognostic factor for breast cancer, and it is known that the prognosis of HER2-positive cases is significantly poor particularly in cases of lymph node metastasis. HER2 is attracting attention as an important factor that determines the indication of trastuzumab and as a predictor of the effect of anticancer agents such as anthracyclines and taxanes.
  • trastuzumab is a genetically engineered HER2 monoclonal antibody that is used as a treatment for recurrent breast cancer.
  • Genentic has derived most of the IgG from humans and only the part that binds to the HER2 receptor from mice.
  • the main action of trastuzumab is to bind to the HER2 receptor and suppress the growth of cancer cells, but it is also said to have an action of destroying cancer cells via NK cells and monocytes.
  • overexpression of HER2 protein is examined by an immunohistochemical method (IHC method), and overexpression of HER2 gene is examined by FISH method.
  • IHC method immunohistochemical method
  • FISH method FISH method
  • first, positive, negative, and border areas are selected by a simple IHC method, and if positive, trastuzumab administration is determined.
  • positive and negative are further selected by the FISH method.
  • the IHC method and the FISH method are compared, the IHC method is simple but has a problem of low accuracy.
  • the FISH method has high accuracy, but the work is complicated and the cost is high. In other words, it is desired to develop a technique that can achieve the same accuracy as the FISH method by the IHC method.
  • the present invention has been made in view of the above problems in the prior art, and an object thereof is to efficiently evaluate an immunohistochemical image.
  • the invention according to claim 1 is a method of staining a tissue section using a staining reagent in which a biological material recognition site is bound to particles encapsulating a fluorescent material, and the stained tissue
  • a tissue evaluation method comprising the steps of:
  • the invention according to claim 2 is a method of staining a tissue section using a staining reagent in which a biological material recognition site is bound to a particle containing a fluorescent substance, and a field of view of 30 mm 2 or more on the stained tissue section. Measuring the number of fluorescent luminescent spots from the microscope image of, and evaluating the expression level of the biological material corresponding to the biological material recognition site based on the measured number of fluorescent luminescent spots. This is a characteristic organization evaluation method.
  • the invention according to claim 3 is a method of staining a tissue section using a staining reagent in which a biological material recognition site is bound to particles encapsulating a fluorescent substance, and a field of view of 300 mm 2 or more on the stained tissue section. Measuring the number of fluorescent luminescent spots from the microscope image of, and evaluating the expression level of the biological material corresponding to the biological material recognition site based on the measured number of fluorescent luminescent spots. This is a characteristic organization evaluation method.
  • immunohistochemical images can be evaluated efficiently.
  • nanoparticles encapsulating a fluorescent substance bound with a biological substance recognition site are used.
  • fluorescent substance examples include organic fluorescent dyes and quantum dots (semiconductor particles). When excited by ultraviolet to near infrared light having a wavelength in the range of 200 to 700 nm, it preferably emits visible to near infrared light having a wavelength in the range of 400 to 900 nm.
  • Organic fluorescent dyes include fluorescein dye molecules, rhodamine dye molecules, Alexa Fluor (Invitrogen) dye molecules, BODIPY (Invitrogen) dye molecules, cascade dye molecules, coumarin dye molecules, and eosin dyes. Examples include molecules, NBD dye molecules, pyrene dye molecules, Texas Red dye molecules, cyanine dye molecules, and the like.
  • quantum dots containing II-VI group compounds, III-V group compounds, or group IV elements as components ("II-VI group quantum dots”, "III-V group quantum dots”, " Or “Group IV quantum dots”). You may use individually or what mixed multiple types.
  • CdSe CdS, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaP, GaAs, Si, and Ge, but are not limited thereto.
  • a quantum dot having the above quantum dot as a core and a shell provided thereon.
  • CdSe / ZnS when the core is CdSe and the shell is ZnS, it is expressed as CdSe / ZnS.
  • CdSe / ZnS, CdS / ZnS, InP / ZnS, InGaP / ZnS, Si / SiO 2 , Si / ZnS, Ge / GeO 2 , Ge / ZnS, and the like can be used, but are not limited thereto.
  • the quantum dots those subjected to surface treatment with an organic polymer or the like may be used as necessary. Examples thereof include CdSe / ZnS having a surface carboxy group (manufactured by Invitrogen), CdSe / ZnS having a surface amino group (manufactured by Invitrogen), and the like.
  • the nanoparticle encapsulating the fluorescent substance means a substance in which the fluorescent substance is dispersed inside the nanoparticle, whether the fluorescent substance and the nanoparticle itself are chemically bonded or not bonded. Good.
  • the material constituting the nanoparticles is not particularly limited, and examples thereof include polystyrene, polylactic acid, and silica.
  • the nanoparticles encapsulating the fluorescent material used in the present invention can be produced by a known method.
  • silica nanoparticles encapsulating an organic fluorescent dye can be synthesized with reference to the synthesis of FITC-encapsulated silica particles described in Langmuir 8, Vol. 2921 (1992).
  • FITC-encapsulated silica particles described in Langmuir 8, Vol. 2921 (1992).
  • Silica nanoparticles encapsulating quantum dots can be synthesized with reference to the synthesis of CdTe-encapsulated silica nanoparticles described in New Journal of Chemistry Vol. 33, p.561 (2009).
  • Polystyrene nanoparticles encapsulating organic fluorescent dyes may be copolymerized using an organic dye having a polymerizable functional group described in US Pat. No. 4,326,008 (1982) or polystyrene described in US Pat. No. 5,326,692 (1992). It can be produced using a method of impregnating nanoparticles with an organic fluorescent dye.
  • Polymer nanoparticles encapsulating quantum dots can be prepared using the method of impregnating polystyrene nanoparticles with quantum dots described in Nature Biotechnology, Vol. 19, page 631 (2001).
  • the average particle diameter is obtained by taking an electron micrograph using a scanning electron microscope (SEM), measuring the cross-sectional area of a sufficient number of particles, and taking each measured value as the area of the circle, the diameter of the circle is the particle diameter As sought. In the present application, the arithmetic average of the particle diameters of 1000 particles is defined as the average particle diameter. The coefficient of variation was also a value calculated from the particle size distribution of 1000 particles.
  • the biological material recognition site is a site that specifically binds and / or reacts with the target biological material.
  • a nucleotide chain, protein, antibody and the like can be mentioned.
  • an anti-HER2 antibody that specifically binds to HER2 which is a protein present on the cell surface
  • an anti-ER antibody that specifically binds to an estrogen receptor (ER) present in the cell nucleus and actin that forms a cytoskeleton
  • An anti-actin antibody that specifically binds to the actin.
  • those in which anti-HER2 antibody and anti-ER antibody are bound to fluorescent substance-encapsulating nanoparticles can be used for selection of breast cancer medication, and are preferable.
  • the mode of binding between the biological substance recognition site and the fluorescent substance-encapsulating nanoparticles is not particularly limited, and examples thereof include covalent bonding, ionic bonding, hydrogen bonding, coordination bonding, physical adsorption, and chemical adsorption.
  • a bond having a strong bonding force such as a covalent bond is preferred from the viewpoint of bond stability.
  • an organic molecule that connects between the biological substance recognition site and the fluorescent substance-containing nanoparticle.
  • a polyethylene glycol chain can be used, and SM (PEG) 12 manufactured by Thermo Scientific can be used.
  • a silane coupling agent that is a compound widely used for bonding an inorganic substance and an organic substance can be used.
  • This silane coupling agent is a compound having an alkoxysilyl group that gives a silanol group by hydrolysis at one end of the molecule and a functional group such as a carboxyl group, an amino group, an epoxy group, an aldehyde group at the other end, Bonding with an inorganic substance through an oxygen atom of the silanol group.
  • silane coupling agent having a polyethylene glycol chain for example, PEG-silane no. SIM6492.7 manufactured by Gelest
  • silane coupling agent you may use 2 or more types together.
  • organic fluorescent dye-encapsulated silica nanoparticles As a reaction procedure between the organic fluorescent dye-encapsulated silica nanoparticles and the silane coupling agent, a known method can be used. For example, the obtained organic fluorescent dye-encapsulated silica nanoparticles are dispersed in pure water, aminopropyltriethoxysilane is added and reacted at room temperature for 12 hours. After completion of the reaction, organic fluorescent dye-encapsulated silica nanoparticles whose surface is modified with an aminopropyl group can be obtained by centrifugation or filtration. Subsequently, by reacting the amino group with the carboxyl group in the antibody, the antibody can be bound to the organic fluorescent dye-encapsulated silica nanoparticles via an amide bond. If necessary, a condensing agent such as EDC (1-Ethyl-3- [3-Dimethylaminopropyl] carbohydrate, Hydrochloride: manufactured by Pierce (registered trademark)) can also be used.
  • a linker compound having a site capable of directly binding to organic fluorescent dye-encapsulated silica nanoparticles modified with organic molecules and a site capable of binding to a molecular target substance can be used.
  • sulfo-SMCC Sulfosuccinimidyl 4 [N-maleidomethyl] -cyclohexane-1-carboxylate: manufactured by Pierce
  • sulfo-SMCC Sulfosuccinimidyl 4 [N-maleidomethyl] -cyclohexane-1-carboxylate: manufactured by Pierce
  • the same procedure can be applied regardless of whether the fluorescent substance is an organic fluorescent dye or a quantum dot. That is, by impregnating a polystyrene nanoparticle having a functional group such as an amino group with an organic fluorescent dye or a quantum dot, a fluorescent substance-containing polystyrene nanoparticle having a functional group can be obtained.
  • EDC or sulfo-SMCC is used. In this way, antibody-bound fluorescent substance-encapsulated polystyrene nanoparticles can be produced.
  • the staining method of the present invention is not limited to a pathological section tissue and can also be applied to cell staining.
  • the method for preparing a section to which the staining method of the present invention can be applied is not particularly limited, and those prepared by a known method can be used.
  • the pathological section is immersed in a container containing xylene to remove paraffin.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, xylene may be exchanged during the immersion.
  • the pathological section is immersed in a container containing ethanol to remove xylene.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. Further, if necessary, ethanol may be exchanged during the immersion.
  • the pathological section is immersed in a container containing water to remove ethanol.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. Moreover, you may exchange water in the middle of immersion as needed.
  • the activation process of the target biological substance is performed according to a known method.
  • the activation conditions are not particularly defined, but as the activation liquid, 0.01 M citrate buffer (pH 6.0), 1 mM EDTA solution (pH 8.0), 5% urea, 0.1 M Tris-HCl buffer, etc. are used. be able to.
  • As the heating device an autoclave, a microwave, a pressure cooker, a water bath, or the like can be used.
  • the temperature is not particularly limited, but can be performed at room temperature. The temperature can be 50-130 ° C. and the time can be 5-30 minutes.
  • the section after the activation treatment is immersed in a container containing PBS (Phosphate Buffered Saline) and washed.
  • PBS Phosphate Buffered Saline
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, the PBS may be replaced during the immersion.
  • each fluorescent substance-encapsulating nanoparticle PBS dispersion may be mixed in advance or separately placed on a pathological section separately. Also good.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the reaction time is preferably 30 minutes or more and 24 hours or less.
  • a known blocking agent such as BSA-containing PBS
  • the stained section is immersed in a container containing PBS, and unreacted fluorescent substance-containing nanoparticles are removed.
  • the temperature is not particularly limited, but can be performed at room temperature.
  • the immersion time is preferably 3 minutes or longer and 30 minutes or shorter. If necessary, the PBS may be replaced during the immersion. Hematoxylin-eosin staining may be performed for tissue morphology observation. A cover glass is placed on the section and sealed. A commercially available encapsulant may be used as necessary.
  • the number of fluorescent bright spots or emission luminance is measured from a microscope image of a wide field of view with respect to a stained pathological section.
  • An excitation light source and a fluorescence detection optical filter corresponding to the absorption maximum wavelength and fluorescence wavelength of the fluorescent substance used are selected.
  • the number of bright spots or emission luminance can be measured by using commercially available image analysis software, for example, all bright spot automatic measurement software G-Count manufactured by Zeonstrom Co., Ltd.
  • the field of view of the microscopic image is preferably 3 mm 2 or more, more preferably 30 mm 2 or more, and further preferably 300 mm 2 or more.
  • the expression level of the target biological substance is evaluated. Specifically, it can be evaluated that the higher the number of bright spots, the higher the expression level of the biological substance. Moreover, it can be evaluated that the higher the emission luminance, the higher the expression level of the biological substance.
  • Step (3) The mixture prepared in Step (1) was added to the mixture prepared in Step (2) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
  • Step (4) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
  • SEM scanning electron microscope
  • Step (2) Quantum dot-encapsulated silica: synthesis of CdSe / ZnS-encapsulated silica nanoparticles having an emission wavelength of 655 nm
  • CdSe / ZnS-encapsulated silica nanoparticles (hereinafter referred to as “nanoparticles 2”) were prepared by the following steps (1) to (5).
  • Step (1) 10 ⁇ L of CdSe / ZnS decane dispersion (Invitrogen Qdot655) and 40 ⁇ L of tetraethoxysilane were mixed.
  • Step (2) 4 mL of ethanol and 1 mL of 14% aqueous ammonia were mixed.
  • Step (3) The mixture prepared in Step (1) was added to the mixture prepared in Step (2) while stirring at room temperature. Stirring was performed for 12 hours from the start of addition.
  • Step (4) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
  • Step (1) 1 mg of nanoparticles 1 was dispersed in 5 mL of pure water. Next, 100 ⁇ L of an aminopropyltriethoxysilane aqueous dispersion was added and stirred at room temperature for 12 hours. Step (2): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Step (3): Ethanol was added to disperse the sediment, followed by centrifugation again.
  • Step (4) The amino group-modified silica nanoparticles obtained in step (3) were adjusted to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid).
  • Step (6) The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed.
  • Step (7) PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing
  • Step (8) When 100 ⁇ g of the anti-HER2 antibody was dissolved in 100 ⁇ L of PBS, 1 M dithiothreitol (DTT) was added and reacted for 30 minutes.
  • Step (10) Using the nanoparticle 1 as a starting material, the particle dispersion obtained in step (7) and the reduced anti-HER2 antibody solution obtained in step (9) are mixed in PBS and allowed to react for 1 hour. It was. Step (11): 4 ⁇ L of 10 mM mercaptoethanol was added to stop the reaction. Step (12): The reaction mixture was centrifuged at 10,000 G for 60 minutes, and the supernatant was removed. Then, PBS containing 2 mM of EDTA was added, the precipitate was dispersed, and centrifuged again. The washing
  • Fluorescent substance-encapsulated silica nanoparticles bound with anti-HER2 antibody obtained from nanoparticle 1 as a starting material are labeled material A, and phosphor-encapsulated silica nanoparticles bound with anti-HER2 antibody obtained from nanoparticle 2 as a starting material. This is labeled material B.
  • an anti-HER2 antibody was bound to Cy5 by the method of the following steps (1) to (7).
  • Step (1) When 100 ⁇ g of the anti-HER2 antibody was dissolved in 100 ⁇ L of PBS, 1M dithiothreitol (DTT) was added and reacted for 30 minutes.
  • Step (2) Excess DTT was removed from the reaction mixture with a gel filtration column to obtain a reduced anti-HER2 antibody solution.
  • DTT dithiothreitol
  • Step (3) 1 mg (0.00126 mmol) of an N-hydroxysuccinimide ester derivative of Cy5 (manufactured by GE Healthcare) was adjusted to 3 nM using PBS containing 2 mM of EDTA (ethylenediaminetetraacetic acid).
  • Step (5) The reaction mixture obtained in step (4) was mixed with the reduced anti-HER2 antibody solution obtained in step (2) in PBS and allowed to react for 1 hour.
  • Step (6) 4 ⁇ L of 10 mM mercaptoethanol was added to stop the reaction.
  • Step (7) Excess mercaptoethanol was removed by a gel filtration column to obtain a reduced anti-HER2 antibody solution (labeling material D) bound to Cy5.
  • a labeling material C is obtained by binding an anti-HER2 antibody to CdSe.
  • tissue staining using fluorescent substance-containing nanoparticles Using the antibody-binding labeling materials A to D produced by the methods of the following steps (1) to (10), immunostaining was performed using adjacent sections of human breast tissue whose FISH score was measured in advance. As a stained section, a tissue array slide (CB-A712) manufactured by Cosmo Bio was used. Twenty-four sections with a FISH score of 1-9 were used.
  • Step (1) The pathological section was immersed in a container containing xylene for 30 minutes. The xylene was changed three times during the process.
  • Step (2) The pathological section was immersed in a container containing ethanol for 30 minutes. The ethanol was changed three times during the process.
  • Step (3) The pathological section was immersed in a container containing water for 30 minutes. The water was changed three times along the way.
  • Step (6) The section after autoclaving was immersed in a container containing PBS for 30 minutes.
  • Step (7) PBS containing 1% BSA was placed on the tissue and left for 1 hour.
  • FIG. 1 shows the number of bright spots measured from microscopic images of a plurality of different visual fields (0.3 mm 2 , 3 mm 2 , 32 mm 2 , 324 mm 2 ) and FISH when using the labeling material A (Cy5 inclusion labeling material). It is a figure which shows the relationship with a score.
  • the value of R 2 shown in the figure is the square value of the correlation coefficient between the number of bright spots and the FISH score.
  • FIG. 2 shows the number of bright spots measured from microscopic images of a plurality of different fields of view (0.3 mm 2 , 3 mm 2 , 32 mm 2 , 324 mm 2 ) when using the labeling material B (CdSe-containing labeling material), and FISH It is a figure which shows the relationship with a score.
  • FIG. 3 shows the number of bright spots measured from microscopic images of a plurality of different visual fields (0.3 mm 2 , 3 mm 2 , 32 mm 2 , 324 mm 2 ) and the FISH score when the labeling material C (CdSe) is used. It is a figure which shows a relationship.
  • FIG. 4 shows the number of bright spots measured from microscopic images of a plurality of different fields of view (0.3 mm 2 , 3 mm 2 , 32 mm 2 , 324 mm 2 ) and the FISH score when the labeling material D (Cy5) is used. It is a figure which shows a relationship.
  • Table 1 and FIG. 5 show the square value (R 2 ) of the correlation coefficient between the number of bright spots and the FISH score measured from the microscopic images of each visual field (observation area) for each of the labeling materials A to D.
  • the square value (R 2 ) of the correlation coefficient between the number of bright spots and the FISH score is 0. 5387. This value is approximately 0.734 when converted to the correlation coefficient R, and it can be said that there is a strong correlation between the number of bright spots and the FISH score.
  • the square value (R 2 ) of the correlation coefficient between the number of bright spots and the FISH score was 0. It was 9887.
  • the field of view is 324 mm 2 , it can be said that the correlation is stronger than when the field of view is 32 mm 2 .
  • the correlation between the number of bright spots and the FISH score is good, and the expression level of HER2 is evaluated based on the number of bright spots.
  • the expression level of a specific biological substance can be evaluated by measuring the number of bright spots from an image with a field of view of 3 mm 2 or more without using a time-consuming method such as the FISH method. It is an effective alternative. Therefore, an immunohistochemical image can be evaluated efficiently.
  • an immunohistochemical image can be efficiently evaluated by measuring the number of bright spots from an image of a visual field of 30 mm 2 or more and evaluating the expression level of a specific biological substance based on the number of bright spots. More preferably, an immunohistochemical image can be efficiently evaluated by measuring the number of bright spots from an image with a field of view of 300 mm 2 or more and evaluating the expression level of a specific biological substance based on the number of bright spots.
  • the labeling materials A and B use particles containing a fluorescent substance and are brighter than the labeling materials C and D using a single fluorescent substance, each point of the bright spot is captured from the image. Easy to measure the number of bright spots with high accuracy.
  • a fluorescent labeling material with high brightness as a labeling material for immunohistochemistry, even in a general-purpose fluorescence microscope system, a labeling material that binds to a target molecule even in a low-magnification image of about 20 times the objective lens Can be recognized.
  • a large area on the tissue section can be observed. As a result, it is considered that the detection and quantitative evaluation of a trace amount of biological material that has been conventionally overlooked are possible.
  • the said Example demonstrated the case where the expression level of HER2 was evaluated based on the number of fluorescent luminescent spots, the light emission brightness
  • the tissue evaluation method according to the present invention may be used in the field of pathological diagnosis for evaluating immunohistochemical images.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Afin d'évaluer efficacement une image immunohistochimique, un procédé d'évaluation de tissu selon l'invention comprend une étape de marquage d'une section de tissu à l'aide d'un réactif de marquage obtenu par la liaison d'un site de reconnaissance de substance biologique à une particule encapsulant une substance fluorescente, une étape de mesure du nombre de points à fluorescence intense à partir d'une image microscopique avec un champ de vision de 3 mm2 ou plus sur la section de tissu marqué, et une étape d'évaluation du niveau d'expression de la substance biologique correspondant au site de reconnaissance de la substance biologique sur la base du nombre mesuré de points à fluorescence intense.
PCT/JP2012/056689 2011-03-16 2012-03-15 Procédé d'évaluation de tissu Ceased WO2012124763A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2013504769A JP5863057B2 (ja) 2011-03-16 2012-03-15 組織評価方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2011-057343 2011-03-16
JP2011057343 2011-03-16

Publications (1)

Publication Number Publication Date
WO2012124763A1 true WO2012124763A1 (fr) 2012-09-20

Family

ID=46830828

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2012/056689 Ceased WO2012124763A1 (fr) 2011-03-16 2012-03-15 Procédé d'évaluation de tissu

Country Status (2)

Country Link
JP (1) JP5863057B2 (fr)
WO (1) WO2012124763A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103116018A (zh) * 2013-01-25 2013-05-22 福州迈新生物技术开发有限公司 一种免疫组化质量控制参照物及质量控制方法
JP2015004552A (ja) * 2013-06-20 2015-01-08 コニカミノルタ株式会社 自動染色処理装置
JP5768945B1 (ja) * 2014-07-11 2015-08-26 コニカミノルタ株式会社 生体物質定量方法、画像処理装置、病理診断支援システム及びプログラム
WO2015159580A1 (fr) * 2014-04-15 2015-10-22 ソニー株式会社 Dispositif de mesure de concentration de biomolécule et procédé de mesure de concentration de biomolécule

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012029752A1 (fr) * 2010-08-31 2012-03-08 コニカミノルタエムジー株式会社 Procédé de détection de substance biologique

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5400297B2 (ja) * 2005-12-27 2014-01-29 古河電気工業株式会社 蛍光ナノシリカ粒子、ナノ蛍光材料、それを用いたバイオチップ及びそのアッセイ法
GB0611857D0 (en) * 2006-06-15 2006-07-26 Council Cent Lab Res Councils Lipoprotein assay
JP4985541B2 (ja) * 2008-05-20 2012-07-25 コニカミノルタエムジー株式会社 ナノ粒子内包シリカ、それを用いた生体物質の標識物質および生体物質の標識方法
JP5451236B2 (ja) * 2009-07-31 2014-03-26 富士フイルム株式会社 検出方法、および該検出方法に用いられる磁性体含有誘電体粒子
WO2013051651A1 (fr) * 2011-10-05 2013-04-11 株式会社日立ハイテクノロジーズ Procédé d'analyse de biomolécules et analyseur de biomolécules

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012029752A1 (fr) * 2010-08-31 2012-03-08 コニカミノルタエムジー株式会社 Procédé de détection de substance biologique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOBU ITO: "Breakthrough of immunofluorescence antibody method : Immunofluorescence electron microscopy by Quantum dot", MICROSCOPY, vol. 40, no. 1, 2005, pages 49 - 51 *
JUN MINAGUCHI: "Transvascular accumulation of Sialyl Lewis X conjugated liposome in inflamed joints of collagen antibody-induced arthritic (CAIA) mice", ARCHIVES OF HISTOLOGY AND CYTOLOGY, vol. 71, no. 3, 2008, pages 195 - 203 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103116018A (zh) * 2013-01-25 2013-05-22 福州迈新生物技术开发有限公司 一种免疫组化质量控制参照物及质量控制方法
CN103116018B (zh) * 2013-01-25 2015-04-15 福州迈新生物技术开发有限公司 一种免疫组化质量控制参照物及质量控制方法
JP2015004552A (ja) * 2013-06-20 2015-01-08 コニカミノルタ株式会社 自動染色処理装置
WO2015159580A1 (fr) * 2014-04-15 2015-10-22 ソニー株式会社 Dispositif de mesure de concentration de biomolécule et procédé de mesure de concentration de biomolécule
JP5768945B1 (ja) * 2014-07-11 2015-08-26 コニカミノルタ株式会社 生体物質定量方法、画像処理装置、病理診断支援システム及びプログラム
WO2016006096A1 (fr) * 2014-07-11 2016-01-14 コニカミノルタ株式会社 Méthode de détermination quantitative de substance biologique, dispositif de traitement d'image, système d'aide au diagnostic pathologique, et programme
US9972087B2 (en) 2014-07-11 2018-05-15 Konica Minolta, Inc. Biological substance quantitative determination method, image processing device, pathological diagnosis support system, and computer readable medium

Also Published As

Publication number Publication date
JP5863057B2 (ja) 2016-02-16
JPWO2012124763A1 (ja) 2014-07-24

Similar Documents

Publication Publication Date Title
JP6112173B2 (ja) 組織染色用蛍光標識体および生体物質検出方法
JP5906623B2 (ja) 生体物質発現レベル評価システム
US11435348B2 (en) Tissue staining method
JP6194882B2 (ja) 生体物質の検出方法
WO2013146841A1 (fr) Processeur d'image médical et programme
JP6168047B2 (ja) 組織染色方法
JP5887823B2 (ja) 組織評価方法
WO2016152244A1 (fr) Procédé et système de détection d'une substance biologique cible
WO2015045962A1 (fr) Procédé pour déterminer la quantité de matière biologique dans une section de tissu
WO2016117466A1 (fr) Procédé de quantification de substance biologique, système d'aide à un diagnostic pathologique et programme
JP5863057B2 (ja) 組織評価方法
JP2012194013A (ja) 免疫組織化学染色方法及び反応試薬

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12757431

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2013504769

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12757431

Country of ref document: EP

Kind code of ref document: A1