EP2059906A2 - Procédé de détermination quantitative de la co-localisation de marqueurs moléculaires dans les coupes de tissu - Google Patents

Procédé de détermination quantitative de la co-localisation de marqueurs moléculaires dans les coupes de tissu

Info

Publication number
EP2059906A2
EP2059906A2 EP07788341A EP07788341A EP2059906A2 EP 2059906 A2 EP2059906 A2 EP 2059906A2 EP 07788341 A EP07788341 A EP 07788341A EP 07788341 A EP07788341 A EP 07788341A EP 2059906 A2 EP2059906 A2 EP 2059906A2
Authority
EP
European Patent Office
Prior art keywords
molecular markers
tissue
tissue section
molecular
colocalization
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.)
Withdrawn
Application number
EP07788341A
Other languages
German (de)
English (en)
Inventor
Ansgar Pommer
Bernd Bonnekoh
Raik BÖCKELMANN
Harald Gollnik
Lars Phillipsen
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.)
Tpa Biotech GmbH
Original Assignee
Skinsystec GmbH
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 Skinsystec GmbH filed Critical Skinsystec GmbH
Publication of EP2059906A2 publication Critical patent/EP2059906A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G06—COMPUTING OR CALCULATING; COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • 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
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism

Definitions

  • the invention relates to a method for the quantitative determination of the colocalization of at least two molecular markers in tissue sections, in particular in skin and mucosal tissue sections.
  • tissue section to be examined is mixed with a solution containing a specific reagent, eg. As a dye or an antibody contains.
  • a specific reagent eg. As a dye or an antibody contains.
  • the tissue section containing the molecular groups marked in this way is replaced by different, mostly optical Investigated methods, wherein a signal distribution pattern reflecting the arrangement and distribution of the molecular group of interest is recorded.
  • Fluorescence microscopy in which the fluorescence of with a fluorophore, for example, fluorescein iso-thiocyanate or phycoerythrin, labeled molecules is measured.
  • a fluorophore for example, fluorescein iso-thiocyanate or phycoerythrin
  • Fluorescence signal distribution pattern recorded which reflects the distribution and arrangement of the labeled molecule in the microscope object.
  • the procedure described above must be repeated frequently using different fluorophores.
  • the fluorescence signal distribution patterns obtained in each case for the different molecular species are subsequently to be evaluated together to determine the correlative distribution of the molecular species studied.
  • a method for automating repeated fluorescence labeling for the purpose of determining the colocalization of molecular species in a cell or tissue sample is described in DE 197 09 348 C2.
  • This automated method termed “automatic multi-epitope ligand mapping method” (MELK)
  • MELK uses a device that includes a pipetting system, a 3D handling system, and an optical measuring system.
  • the automatic pipetting system the cell or tissue sample to be examined will gradually with different incubation solutions, each one or more Fluorophore-conjugated reagents, with a fluorescence distribution pattern being recorded by a fluorescence microscope image acquisition device, such as a CCD camera, for each incubation cycle.
  • the invention is therefore based on the object of specifying a method which also allows reliable quantitative statements on the colocalization of molecular markers in tissue sections, in particular in skin and mucosal tissue sections.
  • Tissue section for each of the at least two molecular markers to represent the respective molecular marker - superimposition of the digitized images into a summation image
  • each pixel is assigned a vector whose components are the quantized molecular marker signals of the at least two molecular markers.
  • an image which represents the signal distribution pattern of the respective molecular marker is first recorded and digitized for each of the at least two molecular markers.
  • the images can be generated by different analysis methods. Fluorescence microscopy has proved to be particularly suitable as an analytical method which has been used successfully for many years in biology and medicine. In this case, fluorophores are used as molecular markers whose fluorescence signals are displayed in a fluorescence image.
  • other methods of analysis may be used to generate the digitized images of the tissue section, such as autoradiography, in which the digitized images are read by reading a radiation detector which registers the radioactive radiation emitted by isotope-labeled molecules, or by digitizing one by the radiation blackened photographic film are generated.
  • the digitized images are then superimposed according to the invention to form a summation image, wherein it depends particularly on a preparation point or pixel-precise overlay to obtain exact information about the relative positions of the individual molecule markers to each other.
  • a preparation point or pixel-precise overlay to obtain exact information about the relative positions of the individual molecule markers to each other.
  • the signals for each of the at least two molecular markers in the summation image are quantized pixel by pixel after the image has been overlaid. This can be done by different types of coding, for example by a Trinär-Codtechnik (Basis 3) or in general by an encoding to the base of a number n.
  • a matrix is finally generated, wherein each pixel of the summation image is assigned a vector whose components correspond to the quantized molecular marker signals of the at least two molecular markers.
  • the standardized orientation may be such that the basal membrane zone (BMZ) of the skin tissue in each of the digitized images is disposed substantially horizontally.
  • the digitized images can be aligned anatomically standardized, so that an alignment of the tissue section itself is not required.
  • each of the images is checked for non-informative contents, in particular with regard to possible artifacts, image areas occupied with non-informative contents are marked and these image areas are removed from all images.
  • image areas occupied with non-informative contents are marked and these image areas are removed from all images.
  • the vectors associated with each pixel are normalized to a defined horizontal width of a Hautgewebesammlungs. This can be done by vectorial addition of the signal vectors of the pixels arranged side by side on the defined horizontal width of the Hautgewebeitess. This takes into account the biological situation that the skin is a nonhomogeneous, stratified epithelial tissue that is permanently differentiating perpendicular to the BMZ line, so that normalization only follows along the BMZ line, ie in the horizontal direction, and not perpendicular to it makes sense.
  • the defined horizontal width of the Hautgewebeites may be for example 100 microns.
  • the analysis of the quantized molecular marker signals represented as a vector for each pixel may relate to the entire tissue section, but it is also possible that colocalization of certain molecular markers, e.g. B. certain T-cell subpopulations, toposelective in certain microcompartments, such as the epidermis, in the blood or lymph vessel system or in the dermis to determine quantitatively.
  • the quantitative determination of the colocalization of the molecular marker can be carried out in at least one focal field of the tissue section.
  • a focus field any, for example, a rectangular, circular or any polygonal area to be determined in the tissue section to be examined. Due to the high automation potential of the method according to the invention, it makes sense to perform this while executing a computer program on a data processing system.
  • the invention is further based on the object of providing a device for the quantitative determination of the colocalization of at least two molecular markers in tissue sections, in particular in skin and mucosal tissue sections, which is composed of commercially available standard components and thus can be realized comparatively inexpensively.
  • the unit for generating digitized images is designed as a fluorescence microscope and comprises a CCD camera.
  • Fig. 1 shows a skin tissue section with a reference line along the basal membrane zone (BMZ)
  • Fig. 2 shows a number of fluorescence images of
  • FIG. 9 is a graphical representation of a bivariate analysis for the quantitative determination of the colocalization of a key molecule marker with each one of the remaining molecular marker and
  • Fig. 1 the microscopic image of a skin tissue section is shown. Visible is the upper layer of the horny layer 1 (stratum corneum) and below the epidermis 2 (epidermis). Also recognizable are the basal membrane zone 3 (BMZ), the dermis 4 (dermis) and the subcutis 5 (subcutis). In the area of the basal membrane zone 3 and the dermis 4 different blood and lymph vessels 6 are shown in cross section.
  • BMZ basal membrane zone 3
  • dermis 4 dermis
  • subcutis 5 subcutis
  • the microscopic image of the skin tissue section is first aligned anatomically standardized in a suitable manner. This is preferably done in such a way that the basement membrane zone 3 runs substantially horizontally in the reference coordinate system of the image acquisition apparatus.
  • the mean course of the basement membrane zone 3 is designated in FIG. 1 as BMZ line.
  • FIG. 2 now shows a total of four digitized images of the skin tissue section from FIG. 1, this being different in the context of a test series with n Molecular markers was added. All images were taken by fluorescence microscopy and show the fluorescence signals of the molecular marker shown in each case as a hatched area. In Fig. 2, these carry the reference numeral 7. Further, in Fig. 2, a phase contrast image of the Hautgewebe4.000s is shown. The inclusion of a phase contrast image facilitates the preparation point exact superposition of the fluorescence images in the context of the method according to the invention. As can also be seen from FIG. 2, all fluorescence images as well as the phase contrast image are already anatomically standardized by the horizontal arrangement of the basal membrane zone.
  • a uniform image area (general information field) is defined for all fluorescence images (the fluorescence signals are not shown in FIG. 3 for the sake of clarity) and the phase contrast image.
  • all the fluorescence images are checked for non-informative contents, in particular artifacts.
  • a technical artifact 8 uniquely detectable by an unnatural fluorescence signal value or a particle is identified in the first fluorescence image.
  • the image area occupied by the artifact 8 is marked and removed from the line through the upper edge of the skin tissue section and a line drawn parallel to it in the area of the subcutaneous tissue and laterally delimited by vertical lines in all fluorescence images.
  • FIG. 5 simultaneously represents the summation image from the individual fluorescence images.
  • the colocalization of the individual molecule markers in the tissue section can already be determined on a qualitative level.
  • FIG. 4 now shows a further essential step of the method according to the invention.
  • the signals of all molecular markers in the summation image are quantized pixel by pixel.
  • they are trinely coded, i. H. into the three categories "not available” (value 0), "weakly available” (value 1) and “strongly present” (value 2).
  • a vector whose dimension corresponds to the number of examined molecular markers and thus to the number of different fluorescence images is formed and arranged in a matrix corresponding to the pixel grid of the digitized images.
  • the components of the vectors formed for each pixel of the pixel grid correspond to the quantized ones
  • Molecular marker signals of the n analyzed molecular markers can also be referred to as Molecular Phenotype (MOLP).
  • MOLP Molecular Phenotype
  • FIG. 4 a 4 ⁇ 4 pixel block and specifically the pixel P 32 (3rd row, 2nd column) is considered concretely. This provides no signal for the molecular marker 1, since the fluorescent signal X recognizably does not protrude sufficiently far into the 4 ⁇ 4 pixel block. The situation is different with the molecular marker 2.
  • the weak signal Y becomes represented by the value 1 in the context of quantization.
  • the signal Z (molecular marker 3) is a strong signal. Accordingly, this is quantized with the value 2.
  • no signal is registered for the molecular marker n (ie quantized value 0).
  • the normalization is preferably carried out by vectorial addition of the signal vectors of juxtaposed pixels on the defined horizontal width of Hautgewebeites, which in the present case is suitably set to 100 microns.
  • the standardization takes into account the biological situation that the skin is a permanently differentiating epithelial tissue perpendicular to the BMZ line, so that a standardization only along the BMZ line, ie. H. makes sense in the horizontal direction.
  • This normalization is also referred to as "normalized number of pixels (NORP) with respect to a defined horizontal skin width".
  • the analysis of the quantized molecular marker signals may relate to the entire tissue section, but it is also possible for a toposelective colocalization of certain molecular markers in certain microcompartments of the skin tissue section. As shown by way of example in FIG. 5, these may be the epidermis 2, blood or lymphatic vessels 6 or also the dermis 4.
  • the colocalization of the analyzed molecular markers can also be determined by analyzing individual focal fields of the tissue section make. These can be of any geometrical shape, for example as a square (FIG. 6a), as circles (FIG. 6b) or else as any surface (FIG. 6d).
  • Fig. 7 shows the examination of the immediate pixel neighborhood to a central pixel.
  • this search strategy referred to as "pixel neighborhood search”
  • the eight neighboring pixels laterally or diagonally adjacent to the central pixel are examined in order to recognize the distribution patterns of the molecular markers.
  • Fig. 8 shows a higher order examination of adjacent pixels for the purpose of detecting molecular marker distribution patterns.
  • the pixels adjacent to the central pixel in the first, second, third or nth order are also analyzed, this being the case in the entire general information field (see FIG Microcompartments (see Fig. 5) or focus fields (see Fig. 6) can take place.
  • the molecular neighborhood of T-cell subpopulations in a tissue context can be mapped and analyzed.
  • the signal vectors for each pixel that have been quantitatively evaluated with different search strategies in different subareas of the examined tissue section can now be used for the most diverse biomathematical Evaluation methods are subjected. For example, a monovariate analysis of the signal vectors can be performed. Due to the practically unlimited number of analysable molecular markers when using the method according to the invention, a previously unachievable gain in information, for example in the immunodermahistological diagnosis of cutaneous lymphoma, is possible with the methods known from the prior art.
  • Figure 9 shows the bivariate analysis of the signal vectors for the purpose of quantifying the colocalization of a key molecular marker with each one of the remaining molecular markers analyzed. This makes possible the analysis of the dual-radial relationship of a key molecular marker, a so-called stroke marker, to the remaining molecular markers of a network. It goes without saying that in addition to monovariate and bivariate analysis of the signal vectors, a multivariate analysis can also be carried out as the most complex form of data evaluation. So can be z. For example, the frequency of a particular lymphocyte subpopulation characterized by the positive appearance of two or more molecular markers within the microcompartment of the dermis can be accurately detected.
  • the analysis of the cytoplasmic environment of the nucleus 9 begins in its center by analysis of the pixels adjacent to a central pixel in the first-order first order (see Fig. 7) and then successively in a higher order (see Fig. 8). This is done e.g. until the molecular marker signal corresponding to the cytoplasm 10 breaks off in the signal vectors associated with the individual pixels.
  • the method according to the invention thus allows the analysis of the absolute and relative spatial position of selected cells in relation to one another or to the extracellular matrix.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Computer Vision & Pattern Recognition (AREA)
  • Theoretical Computer Science (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Image Processing (AREA)

Abstract

L'invention concerne un procédé de détermination quantitative de la co-localisation d'au moins deux marqueurs moléculaires dans des coupes de tissu et en particulier dans des coupes de peau ou de muqueuse. Le procédé selon l'invention est caractérisé par les étapes suivantes: formation d'une image numérisée de la coupe de tissu pour chacun des deux marqueurs moléculaires ou plus en vue de révéler le marqueur moléculaire concerné, superposition des images numérisées en une image-somme, quantification par point d'image du signal des deux marqueurs moléculaires ou plus dans l'image-somme, formation d'une matrice, avec association à chaque point d'image d'un vecteur dont les composants sont les signaux quantifiés des deux marqueurs moléculaires ou plus et orientation normalisée de la matrice de manière spécifique au tissu. L'invention concerne en outre un dispositif de détermination quantitative de la co-localisation d'au moins deux marqueurs moléculaires dans les coupes de tissu et en particulier dans des coupes de peau ou de muqueuse, ainsi que l'utilisation du procédé selon l'invention.
EP07788341A 2006-08-30 2007-08-09 Procédé de détermination quantitative de la co-localisation de marqueurs moléculaires dans les coupes de tissu Withdrawn EP2059906A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200610040513 DE102006040513A1 (de) 2006-08-30 2006-08-30 Verfahren zur quantitativen Bestimmung der Kolokalisation von Molekülmarkern in Gewebeschnitten
PCT/EP2007/058280 WO2008025662A2 (fr) 2006-08-30 2007-08-09 Procédé de détermination quantitative de la co-localisation de marqueurs moléculaires dans les coupes de tissu

Publications (1)

Publication Number Publication Date
EP2059906A2 true EP2059906A2 (fr) 2009-05-20

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EP07788341A Withdrawn EP2059906A2 (fr) 2006-08-30 2007-08-09 Procédé de détermination quantitative de la co-localisation de marqueurs moléculaires dans les coupes de tissu

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EP (1) EP2059906A2 (fr)
DE (1) DE102006040513A1 (fr)
WO (1) WO2008025662A2 (fr)

Families Citing this family (2)

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Publication number Priority date Publication date Assignee Title
CN102369530B (zh) 2009-05-28 2016-05-18 皇家飞利浦电子股份有限公司 用于副作用预后和监测的装置
CN104165873A (zh) * 2014-07-22 2014-11-26 中国科学院植物研究所 一种检测活体植物细胞中两种膜蛋白共定位程度的方法

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Publication number Priority date Publication date Assignee Title
DE19709348C2 (de) * 1996-05-29 1999-07-01 Schubert Walter Dr Md Automatisches Multi-Epitop-Ligand-Kartierungsverfahren
US6294331B1 (en) * 1997-08-08 2001-09-25 The United States Of America As Represented By The Department Of Health And Human Services Methods for assessing genetic and phenotypic markers by simultaneous multicolor visualization of chromogenic dyes using brightfield microscopy and spectral imaging
CA2350692A1 (fr) * 1998-10-29 2000-05-11 Cell Works Diagnostics, Inc. Caracterisation de cellules individuelles par des marqueurs multiples
US6678398B2 (en) * 2000-09-18 2004-01-13 Sti Medical Systems, Inc. Dual mode real-time screening and rapid full-area, selective-spectral, remote imaging and analysis device and process
WO2002079752A2 (fr) * 2001-03-30 2002-10-10 Molecular Diagnostics, Inc Detection de cellules anormales
WO2006075333A2 (fr) * 2005-01-13 2006-07-20 Spectrum Dynamics Llc Reconstruction et analyse d'image pluridimensionnelle pour diagnostic par systeme expert

Non-Patent Citations (1)

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Title
See references of WO2008025662A3 *

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DE102006040513A1 (de) 2008-03-27
WO2008025662A3 (fr) 2008-07-24
WO2008025662A2 (fr) 2008-03-06

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