Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6841—In situ hybridisation

Definitions

• the present invention relates to a method for detecting inter chromosomal imbalance by in situ hybridization of fluorescent probes (FISH) on interphase cell nuclei. It also relates to a system implementing this process.
• FISH fluorescent probes
• Trisomy 21 Down syndrome
• Trisomy 21 is the most recurrent anomaly: approximately 1.7 children in 1000 are affected (Stoll et al, 1998).
• FISH in situ hybridization on fluorescent probes
• probes covering the centromeric region or regions of the long arm of chromosome 21 were used for these studies: probes covering the centromeric region or regions of the long arm of chromosome 21 (cosmids, YAC) (Soloviev et al, 1995, Van Opstad et al, 1995; Pierluigi et al, 1996 ; Steinborn et al, 1996). Partial labeling of chromosome 21 with these probes makes it possible to evaluate the number of copies. It is therefore necessary to carry out a signal count, manual or automatic, to detect the cells having three signals corresponding to chromosome 21.
• the interobserver variability comes from the different criteria for defining a 'spot' (i.e. the fluorescent signal), for example its size, its intensity, its texture, but also from the fact that the spots are often found in different focal planes, along the vertical axis Z in the nucleus.
• the results can therefore differ significantly depending on these criteria, which themselves depend on the preparation of the sample in each laboratory.
• a significant bias is constituted by the establishment of the "cut-off", that is to say the value of the "positivity threshold" from which the sample is considered abnormal (Ruangvutilert et al, 2000) . This value is difficult to establish, since the percentage of nuclei having three signals in the control cases can vary between 10 and 20% of the nuclei analyzed.
• the object of the present invention is to provide an automated detection method which, by its very design, thus eliminates the difficulties associated with reading by a human operator, and provides a solution to the problems concerning the variability of sample preparation, which so far have affected analysis.
• the term in situ hybridization is intended to denote a technique making it possible to identify a DNA (or RNA) sequence by means of a probe of specific sequence homologous to that studied. It is based on the complementarity of nucleotides (A / T, A / U, G / C), it can be carried out under precise physico-chemical conditions on chromosomal, cellular or tissue preparations.
• the result of the in situ hybridization process is the formation of a hybrid between a probe and a target.
• In situ hybridization includes a denaturation step, the actual step, of in situ hybridization of the probe on the target, and a step of detecting the hybrid or the probe.
• the probes are labeled with a fluorophore and the hybridization is revealed by fluorescent labeling.
• This technique allows the simultaneous visualization, on the same preparation, of several probes each revealed by a different fluorophore.
• the measurement of two or more signals corresponding respectively to each probe is advantageously carried out by image cytometry.
• Two probes can for example be revealed respectively by a green fluorochrome and by a red fluorochrome.
• the present approach consists in using chromosome paints or chromosome regions for chromosome 21, on the one hand, and, for example, for chromosome 20 or chromosome 22, on the other hand; chromosomes 20 or 22 being taken as a reference because the hybridization signal obtained with a paint of chromosome 21 is comparable to that obtained with a paint of chromosome 20 or chromosome 22 which are of similar size.
• the reference chromosome is preferably chosen from chromosomes 14 and 15.
• the reference chromosome is preferably chosen among chromosomes 17, 19 and 20.
• the reference chromosome is for example chosen from chromosomes 6, 7, 8, 9, 10, 11, 12.
• the reference chromosome is preferably chosen from chromosomes 19, 20, 21, 22.
• the probes used in the in situ hybridization are chromosomal paint probes.
• the term “chromosomal paint probe” or “chromosome paint” is intended to denote a probe or a composition of probes which is suitable for hybridizing, under hybridization conditions, with a target which comprises a predetermined chromosome of a multi- genome. chromosome. If only a fraction of such a chromosome is present in the sample undergoing such hybridization with such a composition of probes, then this fraction hybridizes and is identified.
• a painted probe can be mixed with a second, a third, etc. to allow the simultaneous marking and detection of two, three, etc. predetermined chromosomes.
• chromosomes or from fragments of chromosomes isolated by chromosomal sorting, by flow cytometry or by microdissection of chromosomes.
• the probes used in the present invention are probes covering the centrometric region ( ⁇ satellite DNA), all or part of a chromosomal arm (telomeric sequences for example TTAGG Chan) or specific for specific DNA sequences repeated d '' one or more chromosomes (classic satellite DNA 1, 2 and 3).
• the chromosomal paint probes are marked with a non-isotopic entity such as luminescent agents, dyes, and others.
• a non-isotopic entity such as luminescent agents, dyes, and others.
• This labeling can be done indirectly by labeling the probes with enzymes, biotin, avidin, steptavidin, digoxygenin, haptens and others which will be revealed by luminescent agents or dyes.
• the luminescent agents according to the excitation energy source, can be classified into radio luminescent, chemiluminescent, bioluminescent and photo-luminescent (including fluorescent and phosphorescent).
• the term "fluorescent" generally refers to the property of a substance (such as a fluorophore) to produce light when excited by an energy source such as ultra-light. purple for example. More preferably, the probes are labeled with a fluorophore.
• the inventors propose to detect an interchromosomal imbalance in any animal or human interphase cells, preferably in fetal cells, originating from amniotic fluid or maternal blood.
• the detection of constitutional or acquired chromosomal imbalances (cancers) can also be done for example in circulating blood cells, in skin cells, in oral cells and more generally in all cell types readily available for carrying out a diagnostic test.
• the inventors propose to detect an interchromosomal imbalance, instead of detecting the number of copies of the chromosome analyzed:
• each probe is revealed by a different fluorochrome (in green and in red);
• the intensities of each probe are measured on a minimum of a few hundred (for example, 500) nuclei, after their recognition by the system, within a population control cell and a population to be tested;
• the calculation of the ratio between the two intensity signals of each of the probes makes it possible, by comparing it to the normal reference case, to determine the presence of at least one supernumerary chromosome (chromosome 21 for example).
• the interchromosomal imbalance is then greater than 1, and 1.5 in the case of a trisomy.
• the calculation of the ratio between the two intensity signals of each of the probes also makes it possible, by comparing it to the normal reference case, to determine the presence of chromosomal monosomy.
• the interchromosomal imbalance would then be 0.5.
• the detection method according to the invention makes it possible to avoid all the drawbacks linked to cell culture, and makes it possible to obtain the result more quickly (in a few days) and on a number of which is both statistically significant and non-binding for cell-poor samples (between 500 and a few thousand cells).
• the FISH technique of in situ hybridization of fluorescent probes has already been implemented in a different method disclosed in the patent application published under the number FR2783253 to detect an intrachromosomal imbalance in cellular nuclei in interphase more precisely. to detect loss, or gain of genetic material (deletion, insertion, amplification, duplication ...) of all or part of a chromosomal arm.
• this detection method two probes marked with two different and respectively specific fluorochromes of the long arm and the short arm of a chromosome is hybridized on this same chromosome.
• the measurement phase comprises a first step of acquisition, in a defined measurement plane, of a given number of fields so as to obtain, for a magnification of given image, at least a predetermined number (several hundred or thousands) of analyzable nuclei.
• the acquisition step further comprises a capture, using successive optical filters with narrow passband, of several images corresponding, for each field of observation, respectively to a plurality of planes corresponding to the wavelengths d '' a plurality of fluorochromes (for example: 4,6-Diamidino-2-phenylindole (DAPI) with blue fluorescence, Fluorescein isothiocyanate (FITC) with green fluorescence and Texas Red TM with red fluorescence) these acquired images and a superposition of said acquired and stored images.
• fluorochromes for example: 4,6-Diamidino-2-phenylindole (DAPI) with blue fluorescence, Fluorescein isothiocyanate (FITC) with green fluorescence and Texas Red TM with red fluorescence
• a system for detecting imbalances in chromosomal domains by in situ hybridization of fluorescent probes on interphase cell nuclei, implementing the method according to the invention, comprising: means for carrying out an in situ hybridization of fluorescent probes on two separate chromosomes,
• the fluorescence microscopy means and the image acquisition means cooperate to acquire, in a defined measurement plane, a given number of fields, so as to obtain, for a given image magnification, at least a predetermined number of analyzable nuclei.
• this further comprises filtering means cooperating with the fluorescence microscopy means and the acquisition means for acquiring several corresponding images, for each field, respectively to a plurality of planes corresponding to the wavelengths of a plurality of fluorochromes (for example:
• the fluorescence microscopy means and the acquisition means can advantageously cooperate to acquire, in the same study plan, images corresponding to a control cell population and images corresponding to a cell population subjected to detection.
• FIG. 1 illustrates the main phases of the imbalance detection method chromosomal according to the invention
• FIG. 2 schematically represents the structure of a detection system according to the invention
• FIG. 3 is a flow chart representative of the detection and quantification operations carried out on the images acquired by the detection system according to the invention.
• the samples to be tested are prepared as for a conventional cytogenetic analysis on a slide, whether these samples are cultured or analyzed directly.
• a well centrifuge allowing punctual spreading on the slide (for example a cytospin TM) is recommended for a flat spreading of the cells. This reduces the number of cells “outside focus "during auto playback. Limited spreading also decreases image acquisition time.
• the cultured amniotic samples are treated according to conventional prenatal diagnostic protocols.
• a pepsin treatment is followed by a treatment with a dissociating medium of the cell clusters.
• control slides can be prepared from normal amniotic liquids or normal fibroblasts according to the same protocol.
• analysis may also include blood samples. In the case of a sample from maternal peripheral blood, the detection of fetal cells is made beforehand.
• In situ hybridization can be carried out according to the usual protocols for chromosomal paints (Truong et al, 1998).
• the slides are first incubated for 15 minutes in a 2SSC saline solution, followed by rinsing with PBS buffer, treatment with pepsin in 0.01 N (4 ⁇ g / ml, SIGMA) HCl for 10 minutes.
• the digestion is stopped by a PBS bath for 5 minutes, the slides are fixed for 10 minutes in Carnoy (Ethanol / Acetic Acid, 3: 1 (V / V)) and the slides are dried in air.
• the probe is then denatured for 10 minutes at 70 ° C in 70% Formamide (FLUKA) / 2SSC at pH 7.
• the target DNA that is to say the nuclei on slides, are denatured under the same conditions for 3 minutes .
• the slides are then rinsed with 2SSC and dehydrated in a series of alcohol baths. Hybridization of the probe is carried out overnight at 37 ° C. The post-hybridization washes are carried out at 72 ° C for 5 minutes in 2SSC buffer.
• the detection of the probes is done by the use of antibodies coupled to different fluorochromes (green and red).
• the slides can be stained immediately in DAPI (1 ⁇ g / ml, Molecular Probes) and mounting in p-phenyl-diamine.
• the image cytometry device 1 implemented in the inter chromosomal imbalance detection system according to the invention, comprises, with reference to FIG. 2, an acquisition part 10 including a fluorescence microscope 2, 3, a black and white CCD 5 camera allowing integration times ranging from 40 ms to 10 s, a device (motorized filter holder wheel or motorized filter holder turret) for automatically changing the excitation and emission light spectra by the fluorescent probes 4 arranged between the lamp and the microscope 3 and, optionally, between the microscope 3 the CCD camera 5, and a sample plate holder motorized in X and Y 13, automatically controlled by the device 1 for sampling systematic, and a control and processing part 14 consisting of a computer comprising, in particular, an image acquisition card.
• an acquisition part 10 including a fluorescence microscope 2, 3, a black and white CCD 5 camera allowing integration times ranging from 40 ms to 10 s
• a device motorized filter holder wheel or motorized filter holder turret
• the fluorescence measurement is broken down into two stages: 1) Acquisition and storage of images, after definition of an analysis region on the slide studied. 2) Analysis of the images and exploitation of the data resulting therefrom.
• the first step consists in acquiring, within the limits of a defined surface, a number of given fields, with the objective allowing, for example, a magnification of 40x, so as to obtain 500 to 1000 analyzable nuclei.
• the images are acquired using a CCD camera using selective interference optical filters.
• For each field three image planes corresponding to the wavelengths of the different fluorochromes (blue, green, red) are stored and superimposed ( Figure 2). Benchmarks are established to prevent the observed fields from being out of focus.
• the slides subjected to detection 11 and the control slide 12 are analyzed under the same conditions (in particular, same integration time and same camera gain).
• the procedure is as follows, with reference to FIG. 3: 1) Segmentation of each image and detection of the nuclei to be analyzed, including a separation of nuclei in clusters, as well as the exclusion of the artefacts by criteria of size and morphology), allowing to establish a mask of the nuclei against staining, for example in DAPI, in which the fluorescence intensities are measured.
• “Scatterplot” two-dimensional representation of the intensity values of the two colors. 7) Automatic determination of the mean and standard deviation of the values of the fluorescence ratio for a population of nuclei chosen by cluster analysis, that is to say by a point cloud analysis method and determination of the centers of gravity of these point clouds.
• the fluorescence ratio is calculated for each sample in the same way and under the same conditions. A trisomy 21 is thus detected when the fluorescence ratio for the sample of the normalized patient compared to the control sample is 1.5.
• the image cytometry device further comprises means for detecting nuclei and quantifying fluorescent signals of multiple wavelengths; - the detection and quantification means are arranged to segment nuclei from an analysis on multiple criteria of shape, size, fluorescence density, usually called morphometric and densitometric criteria, giving rise to the creation of a mask for the 'all the fields of a measurement plan.
• a chromosome 21 - specifies cosmid cocktail for the detection of chromosome 21 aberrations in interphase nuclei. Prenat Diagn 1995, 15: 705-711. Verma L, Macdonald F, Leedham P, MacConachie M, Dhanjai S, Hulten M. Rapid and simple prenatal DNA diagnostics of Down's syndrome. Lancet 1998; 352: 9-12. Wei HJ, Su TH, Chien CL, Tzeng CR. Fluorescence in situ hybridization (FISH) as a method to detect aneuploid cells. Fetal Diagn Ther 1997; 12: 309-312.
• FISH Fluorescence in situ hybridization

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Zoology (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Analytical Chemistry (AREA)
• Biotechnology (AREA)
• Physics & Mathematics (AREA)
• Biophysics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=8865062

Family Applications (1)

Country Status (6)

Families Citing this family (7)

Family Cites Families (8)

• 2001
  • 2001-07-03 FR FR0108803A patent/FR2826977B1/fr not_active Expired - Lifetime
• 2002
  • 2002-07-03 US US10/482,809 patent/US20050042609A1/en not_active Abandoned
  • 2002-07-03 CN CNA028172477A patent/CN1558955A/zh active Pending
  • 2002-07-03 WO PCT/FR2002/002317 patent/WO2003004700A1/fr active Application Filing
  • 2002-07-03 EP EP02782464A patent/EP1415000A1/fr not_active Withdrawn
  • 2002-07-03 JP JP2003510458A patent/JP2004535807A/ja active Pending

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events