Classifications

• • 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
  • G01N1/42—Low-temperature sample treatment, e.g. cryofixation
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T7/00—Image analysis
  • G06T7/0002—Inspection of images, e.g. flaw detection
  • G06T7/0012—Biomedical image inspection
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/04—Devices for withdrawing samples in the solid state, e.g. by cutting
  • G01N1/06—Devices for withdrawing samples in the solid state, e.g. by cutting providing a thin slice, e.g. microtome
• • G—PHYSICS
  • G06—COMPUTING OR CALCULATING; COUNTING
  • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
  • G06T2207/00—Indexing scheme for image analysis or image enhancement
  • G06T2207/30—Subject of image; Context of image processing
  • G06T2207/30004—Biomedical image processing
  • G06T2207/30024—Cell structures in vitro; Tissue sections in vitro

Definitions

• the present invention relates to a method for examining a tissue sample according to the preamble of claim 1.
• a large number of diseases are based on morphologically detectable tissue changes. These diseases include benign and malignant tissue proliferation, cancer, inflammation or neurodegenerative diseases.
• tissue samples are diagnosed using histological methods as standard to diagnose such diseases. These methods are now so well established that they offer a high level of diagnostic certainty for many diseases, and are therefore valid e.g. in tumor diagnostics as the "gold standard”.
• Diseased tissues differ from healthy tissues in certain molecular-biologically detectable sizes, such as, for example, genomic sequences, mRNA sequences, the proteome, the methylation of the genomic DNA, the presence of viral or bacterial nucleic acids, and / or the presence of pathogenic molecules.
• Some disease types are even characterized by characteristic gene sequence patterns, gene expression profiles or DNA methylation patterns, which can be recorded on a molecular biological basis and used to diagnose these diseases.
• microarrays are particularly suitable for this.
• a major problem with the molecular biological examination of a tissue sample is that the results obtained are difficult to interpret. For example, it is conceivable that a certain gene Y is typically overexpressed in a known tumor X. A pure tumor sample would immediately be noticed in the molecular biological examination due to a greatly increased gene expression value. However, if the examined sample contains only a small part of tumor tissue, the gene expression value of the total sample would only be slightly increased and it would be difficult to correctly interpret such a gene expression value. It is also conceivable that a known tumor X 'has a specific gene expression profile Y', that is to say a characteristic pattern of gene expression values of two or more genes. In this case, the contamination of the examined sample by non-tumor tissue would distort the tumor-specific gene expression profile Y 'and thus make a diagnosis impossible.
• a frozen tissue sample is placed in a microtome, where a sequence of cuts is made.
• One or more sections from the section sequence are stained with hematoxylin and eosin and examined histologically. The proportion of tumor cells in the individual sections is determined. If this is over 50%, corresponding cuts from the same cut sequence are used for the molecular biological examination. If the proportion of tumor cells in the section is below 50%, the entire section sequence is discarded.
• the knowledge about the composition of the sample obtained through the preceding histological examination is therefore only used to decide whether the sample fulfills a certain exclusion criterion or not.
• the method mentioned is used in basic research, for example to build up gene expression databases, and uses tissue with relatively large tissue samples.
• tissue bank In the event that the sample to be examined does not meet the exclusion criterion, a new sample is taken from the tissue bank, histologically examined again for its proportion of tumor tissue and then possibly subjected to the molecular biological examination.
• the object of the present invention is to provide a method suitable for diagnosis for examining a tissue sample for pathological tissue components and / or other relevant components and their relationship to one another, in which individual samples are subjected to a histological and a non-morphological analysis examination.
• Demach is a method for examining a tissue sample, its genomic and / or proteomic and / or epigenomic and / or biophysical Properties essentially preserved are provided on diseased tissue.
• sections are made from the tissue sample in the usual way, at least one of which is subjected to a histological-cytological and at least one further to a non-morphological analysis examination.
• non-morphological analysis examination means in particular molecular biological studies.
• molecular biological studies is used predominantly.
• non-morphological analysis examinations are also biophysical examinations, e.g. Method for measuring the redox potential, the pH value, the temperature, the oxygen partial pressure or for the determination of metabolites such as lactate or pyruvate.
• At least the quantitative proportion of the pathological tissue or pathological cells and / or another morphological aspect in the tissue sample is determined from the histological-cytological examination by means of an image processing system and then used as a reference variable for evaluating the results of the non-morphological analysis examination.
• the histological-cytological examination e.g. the proportion of necrotic tissue, inflammatory cells or non-pathological connective tissue is also determined and taken into account when evaluating the results of the non-morphological analysis.
• the quantitative proportion of the diseased tissue or diseased cells can be determined, for example, by first staining a section and then examining it using a computer-aided image processing system, in particular.
• a computer-aided image processing system usually consists of an optical system (e.g. Microscope), an image acquisition system (e.g. CCD camera and image acquisition card), a computer and suitable software. With the help of such a system, a large number of sizes can be determined automatically and reproducibly from histological specimens in a short time.
• At least the quantitative proportion of tumor tissue and / or another morphological aspect in the tissue sample is determined with the image acquisition system by means of the image processing system, which with appropriate histological pretreatment of the sample e.g. by counting stained cells and / or cell nuclei or integrating stained tissue areas (to name just two examples) is reliably possible.
• the assessment is e.g. the quantitative proportion of pathological tissue in the sample is determined by counting the cell nuclei and as a reference value - e.g. in the form of a percentage - noted.
• a reference value e.g. in the form of a percentage - noted.
• the experimentally recorded expression pattern can now be corrected and then compared directly with the patterns stored in the database. Consequently, the gene expression pattern of such samples that have a high proportion of non-pathological tissue can also be meaningfully evaluated and interpreted diagnostically.
• Another variant of the method according to the invention provides that a sample is taken from the tissue sample and that at least one partial sample of the sample is subjected to a histological-cytological and at least one further non-morphological analysis examination.
• samples can also be taken from the tissue sample, at least one of which is subjected to a histological-cytological and at least one further non-morphological analysis examination. With this configuration, it is also conceivable that only partial samples of the individual samples are fed to the various examinations.
• the quantitative proportion of the pathological tissue or pathological cells in the tissue sample or the sample is determined from the histological-cytological examination, and the quantitative proportion determined is used as the reference variable for evaluating the results of the molecular biological examination.
• This variant differs from the aforementioned only in that one or more samples are made instead of cuts. What has been said about the aforementioned variant also applies analogously to the random samples or their subsamples.
• Another preferred embodiment of the method provides for the sample to be taken from a tissue sample taken from a patient. With this method of ex-v / vo random sampling, the random samples or the partial samples can be taken, for example, with the aid of a drill core probe or by aspiration with a fine needle. This has the advantage that the tissue sample is not destroyed and, for example, the tumor edges remain unaffected.
• the scratch preparation obtained in this way can be divided directly into two partial samples, one of which according to the invention can be used for histological-cytological and the other for molecular biological examination.
• a suspension can first be produced from the scratch material obtained, which is then only divided into two partial samples. The latter method ensures a better randomization of the composition of the individual subsamples.
• the scraping material is first washed and centrifuged, the pellet obtained is resuspended and then in a centrifuge again with a density gradient. At least one of the fractions obtained in this way is then divided, and according to the invention a partial sample is then fed to the histological-cytological and the other to the non-morphological analysis examination.
• the scraping material can also be processed using any other suitable enrichment and / or fractionation technique.
• the non-malignant tissue remains on the cut surface of the tissue sample treated by means of the scratching technique while largely preserving its tissue structure, while the areas in which the tumor cell aggregates were previously located are missing and are therefore recognizable as depressions in the supervision.
• the tissue sample can now e.g. be cut in a conventional manner with a microtome.
• the first cut corresponds to the cut sequence of the cut surface of the tissue sample that has been removed by the scratch preparation.
• This cut can e.g. are examined histologically for remaining tumor cell aggregates and thus provide information about the success of the sampling.
• the cut can thus serve as a negative control for the assessment of the examination results of the scratch sample.
• sections of the section sequence consist both of non-malignant tissue and of tumor tissue and, as already described, can therefore, according to the invention, be subjected to a histological-cytological and, on the other hand, a non-morphological analysis examination. It is particularly promising to compare the histological, cytological and molecular biological results obtained with those of the scratch preparation.
• At least two cuts or random samples eg core samples, fine needle aspirates, scratch preparations
• at least two partial samples of a random sample are taken from a tissue sample. sample prepared and a histological-cytological or a non-morphological analysis examination.
• the histological-cytological examination determines at least the quantitative proportion of the pathological tissue or Icranky cells in the tissue sample or the sample and then uses this as a reference for the evaluation of the results of the non-morphological analysis examination.
• the individual cuts, samples and their subsamples are summarized below where it makes sense under the term "subsamples”.
• both cuts and e.g. Core samples are made.
• the sections could be used for the histological-cytological examination and the core samples taken from the same sample could be subjected to a molecular biological examination.
• both cuts and scratch preparations are produced from a tissue sample in one process.
• Embodiments in which different types of obtaining sections, random samples or their partial samples are combined with one another are therefore expressly also to be covered by the present invention.
• An important aspect of the invention is that, in the histological-cytological examination, in addition to the quantitative part, the appearance and / or the distribution pattern of the diseased tissue or Icranky cells or other components in the tissue sample is also determined and the results of the non- morphological analysis examination can be used as a basis.
• Conceivable parameters in this context by the image acquisition system in addition to the cell numbers or areas already mentioned can be determined, for example, the distance of stained cells and cell nuclei to one another or to other structures, in particular, for example, blood vessels, or the appearance of individual cells (outer contour) etc., to name just a few examples.
• all suitable and morphological parameters can be determined that are suitable for the additional qualification or quantification of pathological cells or tissues.
• an appearance that manifests itself in numerous outgrowths of a tumor into the surrounding tissue an indication of an increased malignancy of the tumor.
• Another tumor whose molecular biological characteristics are similar to that of the first tumor, but which has a different appearance, can be less malignant. In such a case, the appearance would provide important clues for the interpretation of the results of the molecular biological investigation. The same applies to the distribution pattern of the pathological tissue or cells in the tissue sample.
• the cuts or the random samples are made directly from the fresh tissue sample. This procedure ensures that the genomic, proteomic and / or epigenomic properties of the sample are best preserved.
• cuts e.g. a vibratome is used.
• the tissue sample is frozen before the cuts or the random samples are made. This procedure also ensures the preservation of the genomic, proteomic and / or epigenomic properties. shade the sample.
• a micro- or a cryotome can be used to produce sections.
• tissue sample may be preserved before the cuts are made or before the samples are taken and then embedded in a suitable medium.
• a suitable medium can e.g. Paraffin or other suitable embedding medium.
• Sections can be obtained in the usual way with a microtome. It is again important that the genomic, proteomic and / or epigenomic properties of the sample are preserved.
• a fine needle aspirate or a scratch sample is first taken from a fresh tissue sample and subjected to a molecular biological analysis.
• the tissue sample could then be frozen and then sections made on the cryotome for histological-cytological examination.
• Embodiments in which different types of pretreatment or the preservation of the tissue sample are combined with one another should therefore also be expressly covered by the present invention.
• the general rule is that all the steps described above must be selected so that they do not impair the nucleic acids (DNA, mRNA), proteins and / or the epigenomic methylation pattern, or impair them as little as possible.
• a particularly preferred embodiment of the method according to the invention can be usefully used in clinical diagnostics.
• the tissue sample to be diagnosed which is taken from the patient, is routinely taken as quickly as possible to a pathological laboratory and there to two samples are split, the second sample being fixed and embedded for later, in-depth histological-cytological assessment, while the first sample is applied directly to a support, frozen and cut on the microtome ("quick cut"). Individual sections are then stained histologically and immediately examined by a pathologist who informs the treating doctor of his diagnosis.
• the preferred embodiment provides that the tissue sample which has been applied to a carrier and frozen immediately after removal from a patient is cut with a microtome, and that, in addition to the cuts which are fed to the histological-cytological examination, other cuts of the same cutting sequence are not -morphological analysis examination.
• the pathologist can therefore base his diagnosis on molecular biological findings and thus increase the reliability of the diagnosis - a point that is of great importance especially in intraoperative diagnosis.
• a major advantage of this additional procedural step is that it can be seamlessly integrated into the routine diagnostic procedure without wasting time.
• a suitable molecular biological examination method such as an array-based mRNA analysis
• examination results can be obtained in a relatively short time, which can even be forwarded to the treating doctor at the same time as the histological findings.
• this additional process step the profitable material that is already generated during the quick cut is used profitably.
• the tissue sample remains on the carrier after the cuts have been made, so that it is available for making new cuts. If a cryotome is used and the procedure is quick, it is ensured that the tissue sample remains frozen during the entire process and can be stored in the freezer again without any significant influence on the temperature. It can also be provided that the sample takes on temperature before or during the cutting and is then frozen again.
• tissue sample has been preserved in another way, e.g., as already described, by embedding it in a suitable medium, it can of course also be provided that it remains on the carrier after the cuts have been made and is available for the new cuts to be made. In this case, freezing in the meantime is not necessary.
• the carrier to which the tissue sample is applied is designed in such a way that it can be introduced into the microtome in a reproducible manner, so that the tissue sample has the same relative orientation to the microtome each time sections are made. This ensures that the later re-examined sample largely corresponds to the previous sample and so the histological-cytological and / or molecular biological findings can be compared directly with each other.
• a sample is usually stained and / or fixed, with the genomic, proteomic and / or epigenomic properties of the sample being impaired. Conversely, a sample is usually homogenized during preparation for molecular biology. All topological information of the sample is lost. The preparation of a tissue sample for one examination makes the sample unusable for the other examination. Different partial samples must therefore be used for the two tests.
• the partial samples are e.g. two immediately adjacent cuts from a Mil rotom cut sequence can generally be assumed to have essentially the same composition. This assumption is also generally accepted in pathological circles.
• the composition of the partial samples differ from one another, for example that the tumor tissue in the histological-cytological examined partial sample has a different quantitative proportion than in the molecular biologically examined subsample. This is the case, for example, when the sequence of cuts covers an area of the tumor edge that runs parallel to the cutting plane.
• a quantitative reference quantity averaged over the histologically-cytologically examined sub-sample would consequently mirror an incorrect tumor tissue portion in the molecular-biologically examined sub-sample and thus lead to an incorrect correction of the experimentally recorded expression pattern.
• the tumor tissue in the histologically-cytologically examined sub-sample has a different appearance or a different distribution pattern than in the molecular-biologically examined sub-sample.
• At least two sections are supplied to the histological-cytological examination, these being selected such that it is ensured that the section or sections supplied to the non-morphological analysis examination are in situ between these sections were arranged.
• the number of cuts used for this is e.g. according to the amount of mRNA required for the analysis.
• the tissue-specific composition of two flanking sections and, on the other hand, the molecular biological characteristics of one or more sections are known that were arranged between the flanking sections in the original tissue sample.
• the quantitative proportion, the appearance and / or the distribution pattern of the abnormal tissue or pathological cells in the section or sections examined by molecular biology can thus be determined reliably. For example, if one flanking section has a tumor tissue fraction of 20% and the other flanking section has a tumor tissue fraction of 80%, the pathologist can assume that the tumor tissue fraction in the sections examined in molecular biology, which were in situ between the flanking sections, in the range between 20 and 80%.
• the average proportion of tumor tissue in the sections examined by molecular biology can also be determined or calculated from the tumor tissue parts of the flanking sections.
• a reference quantity can also be determined in these cases, with which, for example, the experimentally acquired gene expression pattern can be corrected and then compared directly with the patterns contained in the databases.
• the partial samples i.e. random samples or their partial samples
• the method is used in intraoperative clinical-pathological diagnostics.
• the method can also be used in periopertive diagnostics, i.e. before or shortly after an operation.
• a method for the detection of genomic DNA, cDNA, mRNA, the epigenomic methylation pattern, proteins, viral or bacterial nucleic acids or other biomolecules is used in the non-morphological analysis examination.
• Biophysical methods such as e.g. Methods for measuring the redox potential, the pH, the temperature, the oxygen partial pressure or for the determination of metabolites such as lactate or pyruvate can be used.
• the detection of genomic DNA can, for example, reveal somatic mutations that can be correlated with various tissue diseases.
• the detection of cDNA, mRNA or proteins can provide information about a disease-specific gene expression pattern.
• the detection of the epigenomic methylation pattern may provide evidence of a disease-specific gene activation pattern.
• the detection of viral nucleic acids can, for example, provide information about virus-related tissue proliferation, while the detection of bacterial nucleic acids, for example, can enable the diagnosis of tissue changes caused by bacteria.
• Other biomolecules, such as infectious prions can indicate a prion-induced tissue change.
• Other biomolecules can be, for example, nucleic acids or proteins from pathogenic parasites.
• a size is recorded in the context of the non-morphological analysis examination that makes it possible to determine the proportion of pathological tissue and / or other tissue components in the tissue sample, and that in this way determined proportion is also used quantitatively to evaluate the results of the molecular biological analysis.
• Such a size can e.g. is the expression level of a gene which is known to be expressed only in healthy tissue, but not in tumor tissue, and which is also known to be the normal expression level.
• the measure of expression of this gene in the sub-sample examined in terms of molecular biology provides a quantity by means of which one can estimate how high the proportion of non-pathological or pathological tissue in the sample is.
• Such sizes are known in the literature, among others. known as a "surrogate marker".
• the determination of the degree of expression of this gene can thus be used in addition to the knowledge of the tissue composition obtained by the histological-cytological examination of the evaluation of the results of the molecular biological examination. The results of the histological-cytological examination can therefore be verified by determining a surrogate marker.
• such a size can also be a gene expression pattern typical of healthy tissue or an expression ratio known for a specific tumor variant between a tumor-specific gene and a gene which is constantly expressed in healthy as well as in erectile tissue ("housekeeping gene").
• the method according to the invention also makes it possible to detect possible surrogate markers or to validate their meaningfulness.
• Microarrays can e.g. are chips on the surface of which molecular probes are immobilized in a matrix-like arrangement (e.g. antibodies, oligonucleotides or polypeptides), whereby the analytes bind to these probes due to hybridization or immunological reactions and can be detected with a scanner.
• a matrix-like arrangement e.g. antibodies, oligonucleotides or polypeptides
• Suspension arrays can e.g. are latex balls ("microbeads") on the surface of which molecular probes are immobilized (e.g. antibodies, oligonucleotides or polypeptides), whereby the analytes bind to these probes due to hybridization or immunological reactions and can be detected with a particle counter.
• molecular probes e.g. antibodies, oligonucleotides or polypeptides
• microarrays and suspension arrays are suitable both for the detection of nucleotides and for the detection of peptides or proteins.
• the biomolecules to be detected are subjected to a labeling step and / or the nucleic acids to be detected are subjected to an amplification step.
• the labeling step can consist, for example, in that the mRNA molecules present in the partial sample are transcribed into labeled cRNA molecules with the aid of a T7 RNA polymerase and Cy3-labeled ribonucleotides.
• Another example of a labeling step is the labeling of proteins present in the partial sample using fluorescence-labeled antibodies or oligonucleotides. kleotide.
• other marking steps known according to the current or future state of the art are also conceivable.
• the amplification step can e.g. consist of a PCR, an rtPCR or another amplification method according to the current or future state of the art.
• the histological-cytological examination comprises at least one staining step and / or at least one immunohistochemical step and / or t 1 -_./ Yw hybridization step. It can be provided that several sections or core samples are each subjected to different histological-cytological or immunohistochemical examinations in order to record a larger number of parameters. For example, a partial sample is stained with hematoxylin and eosin in order to visualize and quantify the cell nuclei and the frequency of mitoses, while a second partial sample with a tumor-specific antibody (e.g. anti-S-100 against malignant melanoma) and a third partial sample e.g. can be treated by in situ hybridization.
• a tumor-specific antibody e.g. anti-S-100 against malignant melanoma
• a method according to one of the preceding claims is used to set up a tumor database, to develop individualized cancer therapies, to adjust a patient to individualized cancer therapy and / or to answer scientific questions.
• Two tumors of the same type are macroscopically assessed and prepared in a rapid section examination and then frozen on a suitable support, embedded in medium in a freezing microtome.
• Tumor parenchyma Tumor stroma Tumor A 80% 20% Tumor B 50% 50% Table 1: histologically determined tissue composition of the two tumors
• the second, third and fourth section (each with a thickness of 30 ⁇ m) is recorded in a reaction vessel with RNA-later in order to preserve the integrity of the mRNA.
• RNA-later After total RNA extraction with a commercial kit (eg RNeasy from Qiagen), the poly-A RNA is selectively amplified (Wang et al. Nature Biotechnology, April 2001), whereby one or two rounds of amplification can be carried out as required.
• the amplified RNA or cDNA is prepared and can be analyzed by hybridization on a DNA microarray. In this way, all mRNA molecules present in the examined tissue are determined quantitatively.
• a quantitative gene expression profile is thus obtained, which can be compared with standard gene expression profiles of different tissue and tumor types, in order to determine the diagnosed tumor more precisely.
• standard gene expression profiles for a large number of tumor and tissue types are currently being determined in various laboratories worldwide.
• genes A-1 were examined.
• the following gene expression profiles (given in arbitrary units) were obtained for the two tumors examined:
• a comparison of the normalized gene expression profiles with the standard gene expression profiles shows that tumor A is subtype 2 of cancer X, while tumor B is subtype 1 of cancer X.
• the differentiation of the two tumors is possible solely through the array analysis and the subsequent normalization of the gene expression profiles.
• the histological examination only enables the tumor to be identified as carcinoma X, but not the differentiation into the subtypes described.

Landscapes

• Engineering & Computer Science (AREA)
• Physics & Mathematics (AREA)
• Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medical Informatics (AREA)
• Analytical Chemistry (AREA)
• Radiology & Medical Imaging (AREA)
• Quality & Reliability (AREA)
• Computer Vision & Pattern Recognition (AREA)
• Theoretical Computer Science (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Sampling And Sample Adjustment (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=34877205

Family Applications (1)

Country Status (6)

Families Citing this family (1)

Family Cites Families (11)

• 2004
  • 2004-02-26 DE DE102004009934A patent/DE102004009934A1/de not_active Withdrawn
• 2005
  • 2005-02-25 EP EP05715532A patent/EP1718947A1/fr not_active Withdrawn
  • 2005-02-25 JP JP2007500162A patent/JP2007534319A/ja active Pending
  • 2005-02-25 WO PCT/EP2005/001984 patent/WO2005083388A1/fr not_active Ceased
  • 2005-02-25 US US10/590,927 patent/US20070184430A1/en not_active Abandoned
  • 2005-02-25 AT AT05715532T patent/ATE535795T1/de active

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events