Study of fluorescence in situ hybridization for detection of chromosome aberration

The authors applied fluorescence in situ hybridization (FISH) technique for the detection of chromosome aberration in interphase nuclei using the probe specific to alphoid repeats on chromosome 11 and X. Chromosome 11 specific probe showed two major spots in lymphocyte nuclei, while X specific probe showed single spot in male and double spots in female respectively. On the other hand three spots were detected in most of the nuclei from HeLa cells with 11 and X specific probes. We concluded that FISH with the use of chromosome specific probe may become a useful and reliable tool for the detection of chromosome aberration in interphase nuclei.     

Non-isotopic detection of chromosome 1 in human meiosis and demonstration of disomic sperm nuclei

Summary Nonradioactive in situ hybridization with the biotin-labeled chromosome 1-specific probe pUC1.77 was performed on human mitotic and meiotic chromosomes, and on sperm nuclei. The streptavidine-horseradish-peroxidase and diaminobenzidine detection system demonstrated heteromorphisms in the Iq12 heterochromatic region, not only in mitotic cells but also in mature sperm heads. The localization of chromosome 1 could be traced through all meiotic stages and in the sperm nuclei. The frequency of chromosome 1 disomy in human sperm, as indicated by two distinct hybridization signals, was calculated to be 0.41%.     

应用双色荧光原位杂交技术检测克氏综合征

探讨用双色荧光原位杂交技术（dual-color fluorescence in situ hybridization,D-FISH）检测性染色体数目异常克氏综合征的应用价值,建立常规分裂期染色体和间期细胞FISH技术的实验方法。以Biotin标记的X染色体α-卫星DNA（pBamX7）探针和以Digoxigenin标记的Y染色体长臂末端重复序列（pY3.4）探针对19例克氏综合征标本同时进行外周血染色体及其间期细胞核的原位杂交,分别用Avidin-FITC和Rhodamine-FITC及其Anti-avidin进行信号的检测与放大,DAPI复染。于Olympus AX-70型荧光显微镜下,分别通过WIB、WIG及其WU滤光镜观察杂交信号及其染色体或间期核背景,并统计外周血中期染色体及其间期细胞核的杂交信号颗粒数量。在显微镜下可见以Biotin标记的pBamX7探针显示2个绿色杂交信号,以Digoxigenin标记的pY3.4探针显示1个红色杂交信号,染色体或间期核背景经DAPI复染显示蓝色;18例出现XXY杂交信号的细胞,染色体及其间期细胞核杂交平均出现率分别为95.89%和95%,明显大于正常对照标准值2.75%,证实核型为47,XXY,与染色体检测的结果一致;其余1例染色体核型检测为嵌合体,XXY杂交信号细胞出现率为92%,同时检出6.7%的XY杂交信号细胞（>正常对照标准值4.17%）。用FISH 技术检测性染色体数目异常克氏综合征具有快速、敏感度高、信号强、背景低、多色等优点,故FISH 技术在产前诊断检测领域中显示其重要的应用价值和发展前景。 Abstract:The objective of the work is to study the technique of dual-color fluorescence in situ hybridization(D-FISH) and its application value in the diagnosis of sex chromosomal count abnormality Klinefelter syndrome and establish an experimental approach to metaphase chromosome and interphase nucleus FISH technique.Biotin labeled alpha satellite X-chromosome DNA(pBamX7) probe and Digoxigenin labeled Y-chromosome long arm terminal repetitive sequence (pY3.4) probe were hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus in 19 cases of Klinefelter syndrome specimens.After being washed,the slides were treated with Avidin-FITC,Rhodamine-FITC and Anti-avidin,amplified with an additional layer and counter-stained with DAPI in an antifade solution.The hybridization signals,chromosomal or interphase nucleus settings were observed respectively with WIB,WIG and WU filters under fluorescence microscope Olympus AX-70,and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted.It was observed under the microscope that the Biotin labeled pBamX7 probe showed 2 green hybridization signals and that the Digoxigenin labeled pY3.4 probe showed 1 red hybridization signal.Chromosome or interphase nucleus counter-stained with DAPI showed blue.The average signal rate of chromosome and interphase nucleus hybridization was 95.89% and 95% respectively,significantly higher than the normal control (2.75%).Karyotype 47,XXY was confirmed,which agrees with the chromosomal findings.One case showed mosaic nuclei.XXY chromosome hybridization signal rate was 92% and XY hybridization signal rate was 6.7%,higher than the normal control rate of 4.17%.FISH is a valuable technique in diagnosing sex chromosomal count abnormality Klinefelter syndrome with the merits of fast speed,high sensitivity,strong signal,low background and multiple color.Therefore,FISH technique can find wide application and potential in prenatal diagnosis.     

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reliability and efficiency of interphase-fish with alpha-satellite probe for detection of aneuploidy

Early diagnosis is very important in pre- and postnatal diagnosis of Down syndrome. This study examines the use of fluorescence in situ hybridization (FISH) to detect trisomy 21 in interphase nuclei and metaphase chromosome obtained from fifty-four Down syndrome patients with a regular type trisomy 21. Three of them showed six hybridization signals on both interphase nuclei and metaphase spreads instead of five signals corresponding to two chromosomes 13 and three chromosomes 21 although they were cytogenetically trisomy 21. Simultaneous application of probe combination revealed that one of the extra signals of chromosomes 13/21 a-satellite probe was located on chromosome 22 in two cases and one extra signal on chromosomes 15 in one case. In addition, another case showed four hybridization signals on both interphase nuclei and metaphase spreads instead of five signals, indicating deletion of the chromosome specific alpha-satellite DNA sequence of chromosome 13/21. These centromeric sequence changes may have pathological significance in the appearance of aneuploidy because they may be involved in the important centromere function.     

Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes

Repeated DNAs from the constitutive heterochromatin of human chromosomes 1 and 18 were used as probes in nonradioactive in situ hybridization experiments to define specific numerical and structural chromosome aberrations in three human glioma cell lines and one neuroblastoma cell line. The number of spots detected in interphase nuclei of these tumor cell lines and in normal diploid nuclei correlated well with metaphase counts of chromosomes specifically labeled by in situ hybridization. Rapid and reliable assessments of aneuploid chromosome numbers in tumor lines in double hybridization experiments were achieved, and rare cells with bizarre phenotype and chromosome constitution could be evaluated in a given tumor cell population. Even with suboptimal or rare chromosome spreads specific chromosome aberrations were delineated. As more extensive probe sets become available this approach will become increasingly powerful for uncovering various genetic alterations and their progression in tumor cells.     

A chromosome 1-specific DNA library from the domestic pig (Sus scrofa domestica).

Chromosomes were prepared from lymphocytes of a male domestic pig and flow-sorted on a dual-laser FACS. Twenty spots were observed, corresponding to the known pig karyotype of 18 pairs of autosomes plus the X and Y. DNA was isolated from 10,000 copies of the presumed chromosome 1 spot, restricted with Sau3A, ligated into the vector pGEM4z, and PCR amplified using universal primers; the products were then re-ligated into pUC18. After transformation into Escherichia coli, 210,000 independent colonies were obtained, 5% of which contained only vector DNA. The average insert size of the library was 405 bp. Southern blotting revealed that 36% of the clones contained single-copy DNA and that the remainder contained moderately or highly repetitive DNA. Screening with a (CA)n probe revealed that roughly 1% of the clones contained microsatellite sequences. A bulk insert of the library was biotinylated by PCR and used as a probe for chromosomal in situ suppression hybridization to pig chromosomes, which confirmed that the library is specific for chromosome 1. However, sequences from the centromeric and telomeric regions seem to be underrepresented in the library.     

New rapid test for prenatal detection of trisomy 21 (Down's syndrome): preliminary report.

OBJECTIVE--To devise and evaluate a rapid screening method for detecting trisomy 21 (Down''s syndrome) in samples of uncultured amniotic fluid cells. DESIGN--Non-radioactive in situ hybridisation with HY128, a 500,000 base pair yeast artificial chromosome probe specific for chromosome 21. Blinded study of 12 karyotypically normal amniotic fluid samples and eight samples trisomic for chromosome 21. SETTING--Cytogenetic and obstetric services at a tertiary referral centre, Copenhagen. MAIN OUTCOME MEASURES--Time necessary to complete the test. Proportion of cell nuclei containing two and three hybridisation signals in karyotypically normal and abnormal amniotic fluid samples. RESULTS--The test could be completed within three to four days after amniocentesis. In the normal samples a mean of 73% (range 61-82%) of the amniotic cell nuclei showed two hybridisation signals and 6% (0-18%) showed three signals. By contrast, among the trisomic samples 29% (19-38%) of the nuclei exhibited two signals and 48% (31-60%) showed three signals. CONCLUSION--The technique clearly distinguished between normal and trisomic samples. Prenatal diagnosis with in situ hybridisation with chromosome specific probes was fast and may make it possible to screen for selected, aneuploidies. However, the technique is still at a preliminary stage and needs further evaluation and refinement.     

Determination of Y chromosome aneuploidy in human sperm nuclei by nonradioactive in situ hybridization.

Sperm nuclei from eight normal, healthy donors were hybridized in situ with the biotin-labeled Y-specific pHY2.1 DNA probe to evaluate the X:Y ratio, the location of the Y chromosome, and the frequency of Y aneuploidy in human sperm. The streptavidine-horseradish-peroxidase and DAB detection system used permitted the unequivocal identification of sperm heads with zero, one, or two hybridization signals and proved superior to either quinacrine staining or radioactive in situ hybridization. The low incidence of 0.27% of sperm nuclei with two Y chromosomes that was found is similar to the frequency of XYY males among newborns. The average proportions of X- and Y-bearing sperm nuclei were 50.3% and 49.4%, respectively, corresponding to the expected 1:1 ratio. The Y heterochromatin was located in the central part of the nucleus in 58% of the Y-carrying sperm cells.     

Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.     

Codling moth cytogenetics: karyotype, chromosomal location of rDNA, and molecular differentiation of sex chromosomes.

We performed a detailed karyotype analysis in the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), the key pest of pome fruit in the temperate regions of the world. The codling moth karyotype consisted of 2n = 56 chromosomes of a holokinetic type. The chromosomes were classified into 5 groups according to their sizes: extra large (3 pairs), large (3 pairs), medium (15 pairs), small (5 pairs), and dot-like (2 pairs). In pachytene nuclei of both sexes, a curious NOR (nucleolar organizer region) bivalent was observed. It carried 2 nucleoli, each associated with one end of the bivalent. FISH with an 18S ribosomal DNA probe confirmed the presence of 2 clusters of rRNA genes at the opposite ends of the bivalent. In accordance with this finding, 2 homologous NOR chromosomes were identified in mitotic metaphase, each showing hybridization signals at both ends. In highly polyploid somatic nuclei, females showed a large heterochromatin body, the so-called sex chromatin or W chromatin. The heterochromatin body was absent in male nuclei, indicating a WZ/ZZ (female/male) sex chromosome system. In keeping with the sex chromatin status, pachytene oocytes showed a sex chromosome bivalent (WZ) that was easily discernible by its heterochromatic W thread. To study molecular differentiation of the sex chromosomes, we employed genomic in situ hybridization (GISH) and comparative genomic hybridization (CGH). GISH detected the W chromosome by strong binding of the Cy3-labelled, female-derived DNA probe. With CGH, both the Cy3-labelled female-derived probe and Fluor-X labelled male-derived probe evenly bound to the W chromosome. This suggested that the W chromosome is predominantly composed of repetitive DNA sequences occurring scattered in other chromosomes but accumulated in the W chromosome. The demonstrated ways of W chromosome identification will facilitate the development of genetic sexing strains desirable for pest control using the sterile insect technique.     

Human gametes and zygotes studied by nonradioactive in situ hybridization

A nonradioactive in situ hybridization technique was applied to human gametes and abnormally fertilized or developed zygotes. Using haptenized chromosome-specific probes, visualization was obtained using immunocytochemistry to achieve a fluorescent stain on specific hybrids. Using a chromosome 1-specific DNA probe, almost all spermatozoa gave a positive result, i.e., one hybridization signal per cell could be observed. Furthermore, it was possible to identify sperm cells with two spots, suggesting nondisjunction. Two cleavage arrested embryos from different patients showed both: two brightly fluorescent spots and two weaker spots with the same DNA probe. Using a Y-specific DNA probe the percentages of positive spermatozoa from the normal males ranged between 48.1% and 49.1%. In an embryo with four grossly haploid chromosome sets, three fluorescent spots were obtained with the Y-specific DNA probe, indicating the penetration of three spermatozoa.     

用间期荧光原位杂交检测卵巢癌细胞中X染色体数目的异常

为了探讨用荧光原位杂交技术（fluorescence in situ hybridization, FISH）检测卵巢癌细胞中性染色体拷贝数目异常的实验方法及其应用价值,收集18例新鲜卵巢癌组织标本,以Biotin标记的X染色体α-卫星DNA（pBamX7）探针与经处理的标本进行卵巢癌细胞核的原位杂交,分别用Avidin-FITC和Anti-avidin进行信号的检测与放大,PI复染。于Olympus AX-70型荧光显微镜下,通过WIB滤光镜观察杂交信号及其细胞核背景,并统计卵巢癌细胞核中的杂交信号颗粒数量。在显微镜下可见以Biotin标记的pBamX7探针显示绿色杂交信号,细胞核背景经PI复染显示桔红色;发现11/18（61%）卵巢癌标本中X染色体拷贝数增加,其余7例（39%）无拷贝数增加。X染色体拷贝数目增多在卵巢癌中有一定比例的发生频率,其在促进卵巢癌发病及其发展过程中起到某种作用,其意义值得进一步研究。     

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

Summary Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproductbility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.     

In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors

Methods for single- and double-target in situ hybridization (ISH) to, cells isolated from solid transitional cell carcinomas (TCC's) of the urinary bladder are described. Single cell suspensions were prepared from solid tumors of the urinary bladder by mechanical disaggregation and fixed in 70% ethanol. Using two DNA probes specific for the centromeres of chromosomes #1 and #18, ISH procedures were optimized for these samples. Human lymphocytes and cells from the T24 bladder tumor cell line were used as controls. In lymphocyte nuclei and metaphase chromosome spreads, ISH showed two major spots for each of the probes. About 80% of the nuclei from T24 cells showed three spots for both the chromosome #1 and #18 specific probes. When nuclei from TCC's were analyzed, often the number of spots for chromosome #1, and to a lesser extent for chromosome #18, differed from the number expected on basis of flow cytometric ploidy measurements. The double target-ISH method in all cases allowed the correlation of numerical aberrations for chromosomes #1 and #18 in one and the same cell. By such analyses a profound heterogeneity in chromosome number was detected in most tumors. In order to optimize the reproducibility of the method and the interpretation of the ISH-signals, criteria for their analysis have been determined. This procedure can now be applied on a routine basis to solid tumor specimens.     

The distribution of cholinergic neurons in the human central nervous system

Choline acetyltransferase (ChAT), the enzyme responsible for the biosynthesis of acetylcholine, is presently the most specific marker for identifying cholinergic neurons in the central and peripheral nervous systems. The present article reviews immunohistochemical and in situ hybridization studies on the distribution of neurons expressing ChAT in the human central nervous system. Neurons with both immunoreactivity and in situ hybridization signals of ChAT are observed in the basal forebrain (diagonal band of Broca and nucleus basalis of Meynert), striatum (caudate nucleus, putamen and nucleus accumbens), cerebral cortex, mesopontine tegmental nuclei (pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus and parabigeminal nucleus), cranial motor nuclei and spinal motor neurons. The cerebral cortex displays regional and laminal differences in the distribution of neurons with ChAT. The medial septal nucleus and medial habenular nucleus contain immunoreactive neurons for ChAT, which are devoid of ChAT mRNA signals. This is probably because there is a small number of cholinergic neurons with a low level of ChAT gene expression in these nuclei of human. Possible connections and speculated functions of these neurons are briefly summarized.     

Nonisotopic in situ hybridization as a method for nondisjunction studies in human spermatozoa

Human spermatozoa were studied with a nonradioactive in situ hybridization method. Using a chemically modified DNA probe and immunocytochemical reactions for visualization, it was possible to obtain hybridization signals in 31 of 32 semen samples. Positive hybridization reactions, depending on cell accessibility, varied from 40% to over 90% for the different samples. Using a chromosome 1-specific DNA probe, disomy for this chromosome was found in 0.67% of all accessible sperm cells.     

Aneuploidy detection in human sperm nuclei using fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was performed on human interphase sperm nuclei to determine the utility of this technique for aneuploidy detection. Repetitive DNA sequences specific for chromosomes 1, 12 and X were biotinylated and hybridized with mature sperm, which had been treated with cetyltrimethylammonium bromide and dithiothreitol to render them accessible to the probes. Detection of bound probe was accomplished with fluoresceinated avidin and antiavidin. For each of the chromosomes studied, chromosome number was determined by counting the fluorescent signals, representing hybridized regions, within the sperm nuclei. The frequencies for disomy, that is for nuclei containing two signals, for chromosomes 1, 12 and X were 0.06%, 0.04% and 0.03%, respectively. The congruence of these results with those determined by the cross-species hamster oocyte-human sperm assay, and the high efficiency of hybridization indicate that FISH is a sensitive and reliable tool for aneuploidy detection in human sperm.     

Comparative FISH analysis of numerical chromosome 7 abnormalities in 5-μm and 15-μm paraffin-embedded tissue sections from prostatic carcinoma

 Interphase fluorescence in situ hybridization (FISH) was performed on 15-μm-thick paraffin sections from prostatic carcinomas using a chromosome 7-specific α-satellite DNA probe. A confocal laser scanning microscope (CLSM) was used for optical sectioning of the thick sections and reconstruction of 3D images. The number of FISH signals was determined by a gallery of optical sections evaluating only complete nuclei. To investiate the influence of section thickness and truncation and nuclei on scoring results, we compared the FISH data from 15-μm sections with signal counts obtained from 5-μm sections. The latter were evaluated by conventional fluorescence microscopy in the same tumor regions previously defined and marked on the slides. After statistical analysis of spot frequencies in tumor and non-tumorous cells (χ² test), we transferred the signal frequencies into a cytogenetic classification (−7, +7, polysomy 7). Based on this classification, most cases showed more than one chromosome 7 aberration type. Trisomy 7 (+7) became apparent in 15-μm-thick sections in all 19 tumors, polysomy 7 (>3 spots) in 18/19 cases, and monosomy 7 (−7) in 13/19 cases. In 5-μm sections, however, trisomy 7 and polysomy 7 were found in only 7/19 and 13/19 cases, respectively, and monosomy 7 in 7/19 cases. When comparing the classification results of tumor cells of the same tumor regions originating either from 5-μm or 15-μm sections, the following discrepancies were noted: in 15-μm sections exclusively, in 12/19 tumors, trisomy 7 was found; in 6/19 cases, polysomy 7; in 8/19 cases, monosomy 7. The high proportion of cases with tumor nuclei expressing only one hybridization signal of chromosome 7 in 15-μm sections could be confirmed as monosomy 7 in five selected cases by double-hybridization using centromere-specific probes for chromosomes 7 and 12. These results demonstrate that numerical chromosome 7 aberrations are more frequently observed in thick (15-μm) paraffin-embedded tissue sections by evaluating only complete nuclei. The use of routine sections (5-μm) for interphase cytogenetic analyses is compromised by a remarkable underestimation of the real chromosome copy numbers. Accepted: 7 November 1996