[go: up one dir, main page]

WO2002065889A1 - Procede pour examiner des echantillons cellulaires et tissulaires - Google Patents

Procede pour examiner des echantillons cellulaires et tissulaires Download PDF

Info

Publication number
WO2002065889A1
WO2002065889A1 PCT/EP2001/001987 EP0101987W WO02065889A1 WO 2002065889 A1 WO2002065889 A1 WO 2002065889A1 EP 0101987 W EP0101987 W EP 0101987W WO 02065889 A1 WO02065889 A1 WO 02065889A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
dna
cell
sample
tumor
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/EP2001/001987
Other languages
German (de)
English (en)
Inventor
Peter Nehls
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.)
RUBIKON AG
Original Assignee
RUBIKON AG
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 RUBIKON AG filed Critical RUBIKON AG
Priority to PCT/EP2001/001987 priority Critical patent/WO2002065889A1/fr
Publication of WO2002065889A1 publication Critical patent/WO2002065889A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • the present invention relates to a method for examining cell and tissue samples, in which DNA damage is detected in the samples at the individual cell level by an immunocytological assay in order to enable a tumor risk assessment.
  • morphological data can also be taken into account.
  • CIN cervical intraepithelial neoplasia
  • Another serious problem is that the known analyzes of cell and tissue samples misclassify 10 to 40% of all cases, i.e. for example tumor cells are not recognized.
  • the starting point for the development of the method according to the invention was the idea that the formation of mutations in genes related to cancer is a critical step for tumor development.
  • Genes relevant in this context include protoonko genes that are involved in controlling cell division and tumor suppressor genes that inhibit neoplastic cell growth. Mutations, i.e. changes in the sequences of these genes, are associated with the development of tumors.
  • DNA-reactive agents such as chemical ones Carcinogens, ultraviolet radiation, ionizing radiation and reactive oxygen species induced.
  • a common feature of these agents is that they cause structural changes in genomic DNA.
  • the DNA reaction products can then be converted into inheritable mutations in dividing cells. In other words, it is assumed that the mutations critical for tumor development are preceded by DNA damage.
  • WO 93/12258 relates to a method for the diagnostic and prognostic monitoring of genotoxic changes in tissue which result from oxidative modifications of DNA. Specifically, several modified nucleotides have been detected in fish that have been exposed to carcinogens in contaminated water.
  • DNA was isolated from tissue samples and, after chemical modification, analyzed using GC / MS.
  • DNA from tissue samples from breast cancer patients was examined in one example using the method mentioned. While increased concentrations of modified nucleotides were found in the DNA from cancer tissue in some cases, this was not the case with DNA from surgical marginal tissue, even if microscopic changes compared to normal tissue were discernible in this tissue. From these results there is no indication of the possibility of a progression assessment; rather, they lead away from the present invention.
  • the publication does not mention the examination of dysplastic tissues.
  • WO 94/25626 a later application by the same inventor as the WO 93/12258 discussed above, suggests using the cellular redox potential as an indicator of the likelihood of cancer.
  • breast cancer tissue samples it is investigated whether analyzes of combinations of different modified nucleotides or their concentration ratios allow a distinction to be made between cancer tissue and normal tissue.
  • the analysis of the isolated DNA is carried out as in the above publication (that is, not for individual cells). Certain concentration ratios are associated with the cellular redox potential and are considered to be meaningful for the distinction between cancerous tissue and normal tissue. Regardless of the complicated methodology, there is no possibility of progression assessment from this document. Studies of dysplastic tissues are not described.
  • the invention is intended to enable the examination of dysplastic cells for tumor risk assessment.
  • tumor cells Compared to normal cells, tumor cells have increased amounts of damaged DNA, such as oxidatively damaged DNA and especially DNA with 8-oxoguanine. This can be used to identify tumor cells.
  • cells such as dysplastic or precancerous cells
  • dysplastic or precancerous cells can have different levels of damaged DNA, such as oxidatively damaged DNA and especially DNA with 8-oxoguanine.
  • Some such cells have levels of damaged DNA that are about the same as normal cells, while others have levels that are about that of tumor cells (i.e., the biological endpoint) development).
  • tumor cells i.e., the biological endpoint
  • the invention provides a method for examining cell and tissue samples for tumor risk assessment, including the identification of already existing tumor cells, which comprises the following stages:
  • the probability of tumor development also includes the probability that tumor cells already exist.
  • the extent of the DNA modification relative to control cells is preferably determined.
  • the process then comprises the following stages:
  • control cells can come from the same sample; for example, it can be undamaged cells.
  • the control cells can, however, also be present in a separately prepared control preparation.
  • the present invention relates to a method for examining dysplastic cells, in which the cells are subjected to an immunocytological assay for determining the extent of DNA modifications.
  • dysplastic cells can be identified on the basis of morphological features.
  • a typical procedure according to the invention is to subject a sample to an immunocytological assay that is known or suspected of containing dysplastic cells.
  • the sample can be a sample obtained during a cancer screening.
  • the sample may also be known or suspected to contain tumor cells. Typically, nothing is known about the previous exposure to a carcinogen for the samples examined according to the invention.
  • the degree of damage to the DNA is then determined with the immunocytological assay at the individual cell level.
  • the degree of damage to a cell in the examined sample as is typically the case with tumor cells, it is determined according to the invention that the examined cell is a tumor cell or a cell developing into a tumor cell.
  • a low degree of damage as is typically the case in a healthy cell is given, it is determined according to the invention that there is no risk of developing into a tumor cell.
  • the extent (degree) of damage to the DNA can be determined as the relative extent of the damage, for example based on healthy cells in the same sample or on cells in a control preparation.
  • the sample mentioned under (c) and (d) above can be a cell or tissue sample in which there are dysplastic or precancerous cells or which are suspected of containing such cells.
  • Dysplastic cells can be identified based on morphological features, as explained elsewhere. The invention is explained in more detail below.
  • a special feature of the method used according to the invention is that the degree of DNA modification in each individual cell can be determined, that is to say that each cell of the sample, for example a cytological preparation, can be assessed individually.
  • This is made possible by sensitive immuno-analytical techniques for the detection and quantitative determination of defined DNA modifications in the DNA of individual cells, for example in suspension or in tissue samples. These techniques can be used to determine any DNA modification for which a suitable monoclonal or polyclonal antibody is available.
  • This antibody (or a fragment thereof, such as preferably F (ab) 2 , but the use of a largely complete antibody is preferred here) is brought into contact with a suitably treated sample, so that a bond between the antibody and the DNA modification for which it is specific.
  • a DNA modification is a damage or structural change in the DNA. Such a change can be a strand break or the chemical change of one or more reactive groups of the DNA contained in the base, sugar and phosphate parts. A chemical change is to be understood as a reaction product.
  • Reaction products are, for example, alkylation products that are created by the action of alkylating agents, oxidation products that are created by the action of reactive oxygen species, and special add-on products of carcinogens.
  • mutagenic DNA damage ie structural changes which lead to sequence changes in proliferating cells.
  • persistence of DNA modifications ie the amount of DNA modifications present in the cells, which results in the long term from the relationship between the formation of new modifications on the one hand and the repair of existing modifications on the other.
  • DNA modifications are detected which are formed by highly reactive oxygen molecules in the genetic material.
  • oxygen species lead to the formation of DNA oxidation products, which include 8-oxoguanine in particular. Its detection can be carried out using a specific antibody, for example against 8-oxoguanine or 8-oxodeoxyriboguanosine.
  • the oxidative DNA damage in tumor cells and in precancerous cells, which develop into tumor cells is significantly measurably increased and the measurement of the oxidative DNA damage allows tumor cells present in a preparation to be clearly identified identify and predict the conversion of precancerous cells into tumor cells.
  • the antibodies which are used according to the invention for the detection of DNA modifications can be polyclonal or monoclonal antibodies. Methods for producing antibodies are known; such procedures can be used. Fragments of antibodies can also be used, it only being necessary that the fragments have the necessary specific binding properties. Some approaches are described below as examples.
  • a suitable antigen for this purpose, for example, a model compound is chemically synthesized that is representative of the modified base that is to be detected later.
  • the low molecular weight compound obtained in this way is not or only to a small extent immunogenic. It is therefore coupled (conjugated) to a suitable carrier.
  • Proteins for example, such as bovine serum albumin (BSA), casein, thyroglobulin or perforated screw hemocyanin, are suitable as carriers.
  • BSA bovine serum albumin
  • casein casein
  • Antibodies are then obtained using standard methods. It is possible to provide the antibodies with a label, in particular a fluorescent label. The marking is also done using standard methods.
  • Antibodies have also been raised against various other carcinogen-DNA adducts.
  • T. Shirai et al. Toxicol. Lett., Vol. 102/103, pp. 441-446 (1998) polyclonal antibodies against 3,2'-dimethyl-4-aminobiphenyl- and 2-amino-1-methyl-6-phenylimidazo [4, 5- b] pyridine-DNA adducts for immunohistochemical detection of the adducts.
  • Monoclonal antibodies against a specific alkylation product of DNA O s -Ethylguanin
  • F. Seiler et al. Mutat. Res., Vol. 385 (No.
  • the antibodies used have a sufficiently high specificity.
  • the antibodies must therefore bind with high affinity to the DNA modification that is to be detected.
  • the reactivity with normal components of RNA and DNA should be as low as possible. Since, when used for testing in cell or tissue samples, there are also numerous other components from the cells which can lead to a cross-reaction with the antibodies, it is important according to the invention to choose antibodies so that they also, if possible, include these other components have low affinity. The person skilled in the art can determine whether this is the case by routine experiments.
  • the antibody material in question is used in an immunocytological assay for suitable preparations, such as, for example, the control preparations described in more detail below.
  • the level of binding of the antibody material is then determined to determine how the binding changes depending on the level of DNA modifications.
  • An antibody material is then preferably selected for the method according to the invention which shows on the one hand a low background binding and on the other hand a high specific binding, ie a high degree of binding in a preparation with a large content of DNA modifications.
  • polyclonal antibodies in particular, it can make sense to provide additional purification steps in order to separate unsuitable antibodies.
  • affinity column in which the column material carries an antigen with which the antibody which is to be used in the method according to the invention reacts. This can significantly improve the specificity of the material.
  • polyclonal antibodies purified in this way have proven to be particularly advantageous.
  • the antibodies with a suitable specificity are then used according to the invention to examine cell or tissue samples in the context of an immunocytological assay.
  • the line or tissue Specimens originate in particular from suspected or ascertained dysplasia or suspected tissue.
  • the samples can be histological tissue sections, for example. Suitable material can also be obtained by fine needle biopsy or punch biopsy (puncture cytology), by collecting cells that have been detached or rejected by surfaces (exfoliative cytology), or by cells that are present in spontaneously emptied secretions, body cavity fluids or irrigation fluids, or by direct Smear sampling from mucosal surfaces can be obtained. Other biopsy samples can also be used.
  • the immunocytological assay is carried out with a prepared cell or tissue sample.
  • a cell or tissue sample is treated with a suitable preservation solution after removal.
  • exemplary solutions are described in U.S. Patent 5,256,571 (Hurley et al.).
  • a fixation can be done with the help of solvents such as methanol, ethanol, acetone or isopropanol.
  • the cell and core membranes are perforated so that the interior of the cell is accessible to the added antibodies and reagents.
  • the fixed sample is rehydrated with buffer solution before the examination.
  • a gynecological sample which is obtained in the usual way by a cytological / smear, is rinsed into a preservation medium. Blood, mucus and nondiagnostic debris are then broken up in a dispersion step and the sample is thoroughly mixed. Cellular material is then collected on a filter membrane and finally transferred to a slide.
  • the swab sample is also transferred to a preservation fluid.
  • a density gradient centrifugation then takes place later for preparation. This removes blood, mucus, material from inflammatory processes and other disruptive components, and you get a cell sample that is enriched with cells to be used for diagnosis.
  • the cell sample can then be resuspended in a suitable medium and transferred to a slide.
  • the sample preparation therefore preferably comprises filtration steps and / or centrifugation steps (such as gradient or equilibrium centrifugation steps).
  • RNA present in the cells can interfere.
  • it contains components that are also present in unmodified DNA are contained and, depending on the antibody, are bound with a certain affinity and, on the other hand, like the DNA modifications which may be recognized by the antibody.
  • damaged RNA is also broken down.
  • a mild alkali treatment is particularly preferred. Ethanol can also be used. This treatment partially makes the DNA single-stranded so that it is more accessible to the antibodies.
  • the denaturation can also be carried out by the action of heat and / or chemicals, for example formamide. However, care must be taken to ensure that there is essentially no destruction of the DNA or removal of the modifications to be detected. It is further preferred to carry out a treatment with a proteolytic enzyme. The background in the later detection reaction can thus be reduced.
  • treatment of the sample with a protein which occupies non-specific adsorption sites is also preferably carried out.
  • the treatment can be carried out with sera or solutions which contain animal proteins, such as IgG or a serum albumin, such as bovine serum albumin (BSA). The sample is then incubated with the antibody specific for DNA damage.
  • the sample is washed to remove unbound antibodies. If the first antibody bears a label, the DNA damage can now be made visible. Otherwise the incubation takes place with a second antibody which is specific for the first antibody and bears a label. After washing to remove unbound second antibody, the labeling of the second antibody and thus indirectly the DNA damage is then made visible.
  • the exact method of visualization and quantitative determination depends on the type of marking. If a fluorescence marker used, which is preferred according to the invention, the evaluation can expediently be carried out with a digital camera. This enables electronic amplification of the immunofluorescence. The images can then be evaluated using a multi-parameter image analysis program. Suitable devices and suitable software are commercially available. Details of a suitable method can be found, for example, in F. Seiler et al., Carcinogenesis, Vol. 14 (No. 9), pp. 1907-1913 (1993).
  • the amount of total DNA contained in the cells can also be measured by fluorescence.
  • an image analysis can be carried out in the same way as for the determination of the DNA modification by means of fluorescence, but using a different dye.
  • the amount of DNA found can additionally be used in the tumor risk assessment intended according to the invention, including the identification of already existing tumor cells.
  • the amount of DNA modification can be related to the amount of DNA and this size can be used in place of the absolute amount of DNA modification. This procedure can also detect and take into account artifacts caused by the binding of antibodies to material that differs from the DNA modification to be detected (cross-reaction).
  • the immunocytological assay is preferably carried out both for the cell or tissue sample to be examined and for one or more control preparations.
  • two control preparations per 20 samples examined can be analyzed. These are standard samples, the nature of which is known.
  • Control preparations can be made, for example, by exposing cells in culture to varying concentrations of a DNA-modifying agent and fixing the cells for a predetermined period after treatment.
  • a DNA-modifying agent for example, by exposing cells in culture to varying concentrations of a DNA-modifying agent and fixing the cells for a predetermined period after treatment.
  • an oxidizing agent such as H 2 0 2
  • the extent of the DNA modification preferably relative to the total DNA content, can be determined for individual cells of the examined sample.
  • an immunocytological assay for cells that originate from different stages of tumor development can be used to derive threshold values for the extent of the DNA modification, such as, for example, the content of reaction products of reactive oxygen species, in particular 8-oxoguanine.
  • the progression estimate with respect to the sample is then carried out by comparing the measurement results for the sample with the threshold values.
  • a cell or tissue sample which has been examined by the immunocytological assay is additionally subjected to histological or cytological staining.
  • This staining then enables the immunocytological data and thus the extent of the DNA modification to be assigned to individual dysplastic cells which, as usual, are classified by histological or cytological staining.
  • a useful coloring is the Pap coloring. A working specification for this can be found, for example, in the book "Gynecological Cytodiagnostics" by Soost and Baur.
  • the 8-oxoguanine content in the DNA of tumor cells, for example cervical tumor cells is always higher than in the DNA of normal cells, for example normal squamous cells.
  • the fluorescence intensities for dysplastic cells were determined and the values obtained were compared with those for normal and for tumor cells. It was shown that there are moderate and severe dysplasias in which the precancerous cells (CIN I, CIN II and CIN III) have a higher oxidative DNA damage than the normal epithelial cells. However, there are also dysplasias of the same severity in which the precancerous cells have the same low oxidative DNA damage as the normal cells.
  • An exemplary procedure for the present invention with cell-specific determination of oxidative DNA damage using example 8-oxoguanine essentially comprises four steps, namely (1) sample preparation; (2) performing the immunocytological method and quantitative determination of the 8-oxoguanine content in each individual cell of the preparation; (3) cytological staining of the preparation; (4) Combination of the 8-oxoguanine values and the optical characteristics determined after staining for the individual cells.
  • optimal and reproducible sample preparation involves removing unwanted accompanying material, such as mucus, bacteria and cell fragments, and separating the cells before applying them to a slide.
  • the cells attached to the slide are then fixed and treated in order to make the cellular DNA accessible to antibody molecules, as also described above. was explained.
  • the preparation is then treated with a monospecific antibody that can recognize and specifically bind to the 8-oxoguanine molecules present in the DNA.
  • a second, fluorescence-labeled antibody is applied to the preparation, which binds to the fc part of the first antibody.
  • the preparation can then be treated with a DNA-binding fluorescent dye, which allows the DNA content of each cell to be quantified.
  • the evaluation is carried out with a fluorescence microscope, which is equipped with a digital camera for measuring the fluorescence signals.
  • the preparation is scanned step by step and each cell is registered on a data carrier with regard to its position, its 8-0xoguanin content and, if appropriate, its DNA content.
  • the preparation is stained according to Papanicolaou for the cytological examination of the cells it contains, including any dysplasia or tumor cells it contains.
  • the colored specimen is placed under the microscope as before, but this time it is scanned step-by-step in transmitted light. The position of the cells and their specific optical characteristics are then also recorded.
  • the method according to the invention does not depend on a conventional classification of the sample. Rather, the method according to the invention provides additional information that cannot be obtained by conventional cytodiagnostics, for example by Papanicolaou staining. Thereafter, dysplastic cells can often be recognized; a forecast of their further development is not possible. ⁇
  • optical identification features preferably those that are accessible for automatic detection / detection, can be used in addition to the precise identification of the cells, for example as tumor cells, or for prognosis (tumor risk assessment) if there is a correlation between 8-0xoguanin content and cell type. For example, cells that show a certain degree of dysplasia can be assessed.
  • nucleus morphology especially the nucleus size, the nucleus shape and the coloring of the chromatin and in particular the ratio of nucleus size to cell size.
  • the method can also be accelerated by using suitable features for preselection.
  • the method according to the invention can in principle be used for all types of cancer.
  • the method is particularly useful for examining dysplastic cells of the cervix. If dysplasias are found in the context of cervical cancer screening cytology, an objective progression assessment can be made with the method according to the invention. More special Areas of application are dysplasia of the female breast or colon.
  • the method according to the invention is particularly applicable to examinations of the lungs / bronchi (especially examination of expectoration, lung secretions), bladder (especially examination of cells from urine samples), thyroid and malignant effusions. In these cases, especially in the case of bladder and effusions, the identification of existing tumor cells can already be improved considerably in comparison with conventional methods.
  • An immunocytological assay for determining DNA modifications can also be used to determine the individual radiation sensitivity. This is of great importance insofar as in the case of radiation therapy, for example of cancerous diseases, in about 30% of the patients inpatient treatment is required because of damage to healthy radiation-sensitive tissues adjacent to the tumor, with about 2% even causing permanent damage. On the other hand, for optimal tumor control, it would occasionally be desirable to use higher than usual radiation doses.
  • a sample of a healthy tissue that is exposed to radiation during the therapy or of a suitable surrogate tissue is irradiated in vitro after the removal. Then the kinetics of the repair of DNA damage is examined.
  • irradiated tissue or cell material is fixed at various times after the irradiation and then subjected to an immunocytological assay, as has already been described.
  • Radiation sensitivity can now be determined by measuring the temporal decrease in the amount of DNA modifications, in particular the amount of modifications caused by reactive oxygen species, and especially the amount of 8-oxoguanine, and correlating them with standard data.
  • a greater decrease in the amount of modifications up to a certain point in time means better repair capacity and less radiation sensitivity. It is possible to make a comparison with statistical averages. On in this way it is possible to individually determine the total radiation dose and the distribution of the individual doses over time in the case of fractional radiation.
  • An immunocytological assay can also be used to determine whether a certain degree of damage (limit value), for example a certain amount of oxidative DNA modifications and especially a certain amount of 8-oxoguanine, is undershot in a sample at a certain time after irradiation. This information can, for example, be used to determine the time and / or dose for further irradiation.
  • a certain degree of damage for example a certain amount of oxidative DNA modifications and especially a certain amount of 8-oxoguanine
  • the radiation sensitivity of tumor tissue can also be determined in the manner described above. In this case, the decrease in the amount of DNA modifications in a tumor tissue sample after radiation is found. This makes it possible to estimate the radiation dose required for treatment.
  • the present invention also includes the provision of a test kit.
  • a test kit comprises one or more components which are required for carrying out the method for examining cell and tissue samples or for examining the sensitivity to radiation. These components have already been discussed above in the explanation of the methods.
  • the test kit comprises an antibody which is specifically directed against a DNA modification, for example against a DNA modification (oxidation product) formed by reactive oxygen species and especially against 8-oxoguanine. It can also is an antibody fragment.
  • This antibody can carry a label, preferably a fluorescent label. If the antibody is not labeled, the test kit preferably contains a second antibody (or a fragment) which has a label, in particular a fluorescent label, and specifically names the first antibody.
  • the test kit may further include one or more of the following components that can be used in the immunocytological assay, as discussed above: RNAse; DNA denaturing agents; protolytic enzyme; Protein for binding unspecific adsorption sites.
  • the test kit also preferably contains buffers and / or solvents used to carry out the method.
  • a cell preservative and / or a fixative may also be included.
  • a test kit for carrying out a preferred embodiment of the method according to the invention, in which the total amount of DNA is determined further comprises a dye for staining the DNA, as has also already been discussed.
  • test kit according to the invention is typically associated with instructions relating to the execution of the method according to the invention.
  • Rabbits were injected subcutaneously and intramuscularly with emulsions containing 600 ⁇ g conjugate and ABM-ZK adjuvant (Linaris GmbH, Bettingen, Germany). The injections were repeated 4, 10 and 22 weeks after the initial immunization. Serum was collected 12 days after the last injection. Antibodies were precipitated with solid ammonium sulfate (50% saturation), dialyzed and stored at -80 ° C. Antibodies with a specificity against 7, 8-dihydro-8-oxo-2 '-deoxyguanosine were obtained with both haptens.
  • tissue sections were fixed in methanol for 15 minutes and rehydrated in 2xSSC (300 mM sodium chloride, 30 mM sodium citrate, pH 7.2). After treatment with RNAse A (200 ⁇ g / ml 2xSSC) and RNAse TI (50 U / ml 2xSSC) for 1 hour at 37 ° C., the sections were washed in 0.14 M NaCl. Cellular DNA was denatured by incubation in a solution containing 70 mM NaOH, 40% EtOH and 0.14 M NaCl for 5 minutes at 4 ° C.
  • 2xSSC 300 mM sodium chloride, 30 mM sodium citrate, pH 7.2
  • RNAse A 200 ⁇ g / ml 2xSSC
  • RNAse TI 50 U / ml 2xSSC
  • the DNA was additionally stained with 0.3 ⁇ M 4, 6-diamidino-2-phenylindole in PBS. To reduce fading, the samples were placed in PBS at pH 8.2 containing 20% glycerin, 10% elvanol and 0.03 M dithiothreitol.
  • a standard fluorescence microscope (Neofluar 40 / 0.75; Zeiss, Oberkochen, Germany) was prepared for the epifluorescence and equipped with a suitable light source (HBO 100 W mercury lamp).
  • Standard filter number 02 (Zeiss) was used for the excitation (maximum: 350 nm) and emission (maximum: 475 nm) of the 4,6-diamidino-2-phenylindole fluorescence spectrum in combination with filter number 14 (Zeiss) for the excitation (maximum : 550 nm) and emission (maximum: 580 nm) of rhodamine isothiocyanate fluorescence.
  • Fluorescence signals were amplified with a single-stage image intensifier (Proxifier BV2532 EG35; Proxitronic, Rothstadt, Germany) and a CCD video camera (Hamamatsu, Japan), the connection being made via fiber optics.
  • the amplified images were fed to a multi-parameter image analysis system (Cytometry Analysis System ACAS; Ahrens, Bargteheide, Germany). This system allowed the integration of images with low signal-to-noise ratios and the quantitative determination of both the fluorescence specific for the labeled antibody and the fluorescence specific for the DNA from the same cell nucleus.
  • Example 3 8-Oxoguani levels in normal and cancerous human cervical cells
  • This example shows that tumor cells have a higher 8-oxoguanine content in genomic DNA than normal cells from the same patient.
  • smears were examined from 9 women who had been diagnosed with cervical carcinoma.
  • the swabs were prepared as explained above and then subjected to an immunocytological assay essentially in accordance with the procedure also described above. An antibody against 8-oxoguanine was used.
  • Example 3 Using a procedure similar to that in Example 3, cervical smears were subjected to an immunocytological test. The content of 8-oxoguanine in each cell of the respective preparation was in the form of fluorescence signals quantitatively determined • (image analysis).
  • the samples were cytologically stained on the basis of the Papanicolaou staining protocol, with an extension of the incubation time in Harris hematoxylin solution and a shortening of the subsequent washing steps being chosen to improve the staining.
  • the evaluation was now carried out in such a way that the fluorescence and color images were compared for each sample. If dysplastic or cancerous cells were identified in the color image on the basis of their optical distinguishing features, a corresponding cytological classification was carried out. In addition, the intensity was determined from the fluorescence image as a measure of the 8-oxoguanine content. Normal epithelial cells served as controls.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour examiner des échantillons cellulaires et tissulaires, afin d'évaluer des risques tumoraux et d'identifier des cellules tumorales déjà existantes, ce procédé consistant en: (a) un test immunocytologique sur un échantillon cellulaire ou tissulaire pour déterminer l'étendue des modifications d'ADN; (b) une évaluation du risque tumoral par comparaison du résultat obtenu en (a) avec une corrélation entre l'étendue des modifications d'ADN et la probabilité d'un développement tumoral. Ce procédé permet en particulier d'examiner un échantillon dont on sait ou suppose qu'il contient des cellules dysplasiques.
PCT/EP2001/001987 2001-02-21 2001-02-21 Procede pour examiner des echantillons cellulaires et tissulaires Ceased WO2002065889A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/EP2001/001987 WO2002065889A1 (fr) 2001-02-21 2001-02-21 Procede pour examiner des echantillons cellulaires et tissulaires

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/EP2001/001987 WO2002065889A1 (fr) 2001-02-21 2001-02-21 Procede pour examiner des echantillons cellulaires et tissulaires

Publications (1)

Publication Number Publication Date
WO2002065889A1 true WO2002065889A1 (fr) 2002-08-29

Family

ID=8164306

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2001/001987 Ceased WO2002065889A1 (fr) 2001-02-21 2001-02-21 Procede pour examiner des echantillons cellulaires et tissulaires

Country Status (1)

Country Link
WO (1) WO2002065889A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993012258A1 (fr) * 1991-12-13 1993-06-24 Pacific Northwest Research Foundation Biomarqueurs du cancer et de lesions genotoxiques obtenus a partir de l'adn
WO1994025626A1 (fr) * 1993-04-30 1994-11-10 Pacific Northwest Research Foundation Profiles d'adn indicateurs du potentiel cellulaire d'oxyreduction et du risque de cancer
WO1996038588A1 (fr) * 1995-05-31 1996-12-05 University Of Leicester Agents analytiques et therapeutiques
WO1997011371A1 (fr) * 1995-09-19 1997-03-27 Cytochem, Inc. Detection et quantification de la 8-hydroxy-adenine avec des anticorps monoclonaux
DE19850680A1 (de) * 1998-11-03 2000-05-04 Peter Nehls Verfahren zur Bestimmung der Reparaturkapazität von DNS-Reparaturenzyme enthaltenden Lösungen und zum Nachweis von DNS-Strukturmodifikationen und Basenfehlpaarungen

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993012258A1 (fr) * 1991-12-13 1993-06-24 Pacific Northwest Research Foundation Biomarqueurs du cancer et de lesions genotoxiques obtenus a partir de l'adn
WO1994025626A1 (fr) * 1993-04-30 1994-11-10 Pacific Northwest Research Foundation Profiles d'adn indicateurs du potentiel cellulaire d'oxyreduction et du risque de cancer
WO1996038588A1 (fr) * 1995-05-31 1996-12-05 University Of Leicester Agents analytiques et therapeutiques
WO1997011371A1 (fr) * 1995-09-19 1997-03-27 Cytochem, Inc. Detection et quantification de la 8-hydroxy-adenine avec des anticorps monoclonaux
DE19850680A1 (de) * 1998-11-03 2000-05-04 Peter Nehls Verfahren zur Bestimmung der Reparaturkapazität von DNS-Reparaturenzyme enthaltenden Lösungen und zum Nachweis von DNS-Strukturmodifikationen und Basenfehlpaarungen

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
COLLINS A ET AL: "PROBLEMS IN THE MEASUREMENT OF 8-OXOGUANINE IN HUMAN DNA. REPORT OFA WORKSHOP, DNA OXIDATION, HELD IN ABERDEEN, UK, 19-21 JANUARY, 1997", CARCINOGENESIS, XX, XX, vol. 18, no. 9, September 1997 (1997-09-01), pages 1833 - 1836, XP000978677 *
DATABASE PUBMED [online] NCBI; NOWAK S ET AL.: "8-oxoguanosine as a marker of neoplastic process in brain", XP002180429, Database accession no. 10791036 *
MALINS D C: "Identification of hydroxyl radical-induced lesions in DNA base structure: Biomarkers with a putative link to cancer development", JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, XX, XX, vol. 40, 1993, pages 247 - 261, XP002086946, ISSN: 0098-4108 *
NEHLS P ET AL: "FORMATION AND PERSISTENCE OF 8-OXOGUANINE IN RAT LUNG CELLS AS AN IMPORTANT DETERMINANT FOR TUMOR FORMATION FOLLOWING PARTICLE EXPOSURE", ENVIRONMENTAL HEALTH PERSPECTIVES, XX, XX, vol. 105, no. SUPPL 5, September 1997 (1997-09-01), pages 1291 - 1296, XP000890074, ISSN: 0091-6765 *
NEUROLOGIA I NEUROCHIRURGIA POLSKA, vol. 33, no. 6, November 1999 (1999-11-01), PL, pages 1339 - 1348 *

Similar Documents

Publication Publication Date Title
DE60200248T2 (de) Verfahren für Lösung-basierte Diagnose
EP1217377B1 (fr) Procédé pour la détection de cellules tumorales et leurs précurseurs dans des frottis du col de l'utérus par la mesure simultanée d'au moins deux marqueurs moléculaires différents
DE69427131T2 (de) Siebtest zur früherkennung von kolorektalen neoplasien
DE2915082C3 (de) Verfahren und Verwendung eines Reagenzsatzes zum Nachweis und zur Charakterisierung von Nukleinsäuren und ihren Sequenzen
DE68925058T2 (de) Verfahren zum frühzeitigen Nachweis von Lungenkrebs
WO2003023057A2 (fr) Procede et kit de diagnostic destines a la selection et/ou detection qualitative et/ou quantitative de cellules
DE3109252C2 (de) Verfahren und Zusammensetzung zur Bestimmung einzelner Leukozyten durch metachromatische Farbstoffdifferentialabsorption
DE60104392T2 (de) Verfahren zum nachweis der effektivität einer krebstherapie
EP1262776B1 (fr) Procédé pour la détermination quantitative de cellules tumorales épithéliales vivantes dans un fluide corporel
DE3650416T2 (de) Zelluläres verfahren zur bestimmung des originalen gewebes.
DE69230890T2 (de) Bestimmung von nukleären matrixproteinen in flüssigkeiten
DE3304795A1 (de) Verfahren zur differentiellen bestimmung der entwicklungsstufen neutrophiler, granulozytischer zellen und anderer leukozyten mittels metachromatischer frabstoffadsorption und einer fluoreszenzlicht-emissionseinrichtung
DE60318415T2 (de) Verfahren zur unterscheidung von metaplasien von neoplastischen oder präneoplastischen läsionen
EP0360822A1 (fr) Procede d'examen de fluides biologiques acellulaires afin d'y depister des transcripts oncogenes cellulaires ou des fragments de ceux-ci
DE69737657T2 (de) Nachweis der alkoholexposition von zellen
DE3883652T2 (de) Verfahren zum sortieren von zellen.
KR102828676B1 (ko) 세포 또는 세포핵의 풍부화 방법
DE3047654A1 (de) Verfahren zum immunologischen nachweis von krebs in menschlichen gewebeschnitten, abstrichen und abblaetternden zytologischen praeparaten sowie dabei verwendete reagentien und diagnosemittel
DE3786175T2 (de) Differentialbindung von potentialsempfindlichen materialien durch lymphocyten.
EP0267355B1 (fr) Procédé d'identification de cytosqueletteprotéines insolubles, spécifiques pour les tissus
DE69714778T2 (de) Immunohistochemischer nachweis des proliferativen zustandes von karzinoma
DE69321454T2 (de) Zytodiagnostisches verfahren unter verwendung von alstonin als selektivem marker und diesen marker enthaltender diagnostischer kit
WO2002065889A1 (fr) Procede pour examiner des echantillons cellulaires et tissulaires
EP1314038B1 (fr) Procede pour l'analyse d'echantillons de selles a des fins diagnostiques
EP0267356B1 (fr) Procédé et moyen de test de détection de lésions de cellules

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP