Detection of HBV-DNA by in situ hybridization using a biotin-labeled probe

A biotin-labeled DNA probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue of 20 patients; 16 were chronic carriers of hepatitis B surface antigen (HBsAg) and 4 had no markers of HBV infection. HBV-DNA was also analyzed in the serum and the liver of these patients by spot and Southern blot hybridization, respectively. Liver specimens from six carriers were positive for HBV-DNA both by in situ and Southern blot hybridization; ten carriers were negative by in situ hybridization, and two of these were positive by Southern blot technique. The staining was granular, mainly cytoplasmic, limited to liver specimens containing replicative forms of HBV-DNA, and associated with detection of HBcAg in hepatocytes by immunofluorescence. The sensitivity of this technique was not sufficient to detect few copies of integrated HBV-DNA. The hybridization procedure was specific, as results were constantly negative in liver specimens of patients without markers of HBV infection, and no reaction was observed using DNA probes lacking HBV-DNA sequences. Detection of HBV-DNA by in situ hybridization, using a biotinylated probe, is a rapid, reproducible, and specific histochemical method. Currently available biotinylated probes are advantageous when absolute sensitivity is not the limiting factor, and they also facilitate studies of the cellular and subcellular distribution of HBV nucleic acids.     

A double labeling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues

This report describes a combined immunocytochemical and in situ hybridization procedure which allows visualization of cellular or viral antigens and viral RNA in the same cell. Cultures infected with visna or measles virus were fixed in periodate-lysine-paraformaldehyde-glutaraldehyde, stained by the avidin-biotin-peroxidase technique using antibodies to viral or cellular proteins and then incubated with radiolabeled specific DNA probes (in situ hybridization). The immunoperoxidase stain was preserved through the hybridization procedure. Nonspecific 'sticking' of probes over peroxidase stained cells was prevented by incorporation of 0.1% Triton X-100 into the hybridization solution and the post-hybridization washes. The in situ hybridization signal (silver grains/cell) on peroxidase-stained cells was reduced relative to hybridization with unstained cells. The double labeling technique was also applied to sections of paraffin-embedded tissues from a sheep infected with visna virus and mice infected with the HNT strain of measles virus. Visna virus RNA was detected in immunocytochemically identified macrophages in the synovium. A greater number of these cells had viral RNA than had viral protein. In measles virus-infected brains viral RNA was detected only in cells with viral protein. This technique provides a new approach to the study of viral pathogenesis by: identifying the types of cells which are infected in the host and identifying points of blockade in the virus life cycle during persistent infections.     

Chromogenic and fluorescent in situ hybridization in breast cancer

Fluorescent (FISH) and chromogenic (CISH) in situ hybridization have recently become part of the diagnostic armamentarium of breast pathologists. HER2 gene testing by FISH and/or CISH has become an integral part of the diagnostic workup for patients with breast cancer. In this era of high throughput technologies, these techniques have proven instrumental for the validation of results from microarray-based comparative genomic hybridization and for the identification of novel oncogenes and tumor suppressor genes. Furthermore, FISH and CISH applied to tissue microarrays have expedited the characterization of genomic changes associated with specific breast cancer molecular subtypes and the identification of novel prognostic and predictive markers. In this review, we provide in this review a critical assessment of CISH and FISH and the impact of the analysis of amplification of specific oncogenes (eg, HER2, EGFR, MYC, CCND1, and FGFR1) and deletion of tumor suppressor genes (eg, BRCA1 and BRCA2) on our understanding of breast cancer.     

用荧光原位杂交技术快速诊断100例早期自然流产胚胎染色体数目异常

目的探讨荧光原位杂交(FISH)技术用于诊断绒毛间期细胞染色体数目异常的临床应用价值。方法采用FISH技术对我院100例50-84天的流产绒毛进行7条染色体(13、16、18、21、22、X和Y)的快速检测。同时,将绒毛接种、培养,进行常规细胞染色体核型分析,作为FISH检测结果的对照。结果被检测的100例样本中,用FISH检测,均获得诊断结果,检测成功率为100%,而常规细胞染色体核型分析,则只有91例获得诊断结果,检测成功率为91%。FISH检测结果与常规细胞染色体核型分析结果均相符合。结论应用FISH技术检测未培养绒毛间期细胞染色体数目异常,具有快速,简便,使用样本量少等优势,具有一定的临床应用价值。     

生物素标记探针原位杂交法检测细胞中c-myc基因的表达

本文采用生物素标记的c-mye基因作探针，通过细胞原位分子杂交技术，对人舌癌、膀胱癌和正常细胞中c-myc基因的转录量进行了定性和半定量分析。结果表明与正常细胞相比，舌癌和膀胱癌细胞中c-myc基因异常高度表达。     

Molecular identification of a small supernumerary marker chromosome by in situ hybridization: diagnosis of an isochromosome 18p with probe L1.84

A dysmorphic child was found by cytogenetic analysis to have an extra small marker chromosome. The marker chromosome was shown to possess a chromosome 18 centromere by in situ hybridization, and probably represents an isochromosome 18p. Centromere specific probes should be of value in identifying extra small marker chromosomes, and thereby provide better understanding of the clinical significance of these.     

Detection and localization of an extra HLA locus in a karyotypically normal male by chromosomal in situ hybridization

The codominant expression of three HLA haplotypes was found in a healthy 21-year-old Black male, whose prometaphase karyotype was normal by light microscopy. He was the sibling of an antenatally diagnosed female fetus with a partial duplication of 6p. The duplication arose from a complex presumably balanced maternal chromosome rearrangement: 46,XX,dir ins(14;6)(14pter----14p11::6p22----6p21.1::14 p11----14qter; 6pter----6p22::6p21.1----6qter). Chromosomal in situ hybridization using a tritium-labeled genomic clone corresponding to a class I HLA gene revealed two sites of hybridization: at 6p21.3, the band to which this probe has been assigned in normal individuals (Morton et al. 1984a) and a second site at 6p11. We postulate that a recombinational event during meiotic pairing in the mother led to the reintroduction into the normal chromosome 6 homolog of a small segment of the original insertion in chromosome 14 which contained the HLA-A and -B determinants.     

G-显带技术、荧光原位杂交和比较基因组杂交技术在产前诊断中的应用

目的 探讨G显带、荧光原位杂交(fluorescencein situ hybridization,FISH)和比较基因组杂交(comparative genomic hybridization,CGH)技术在产前诊断中应用的程序及意义.方法 采集102例妊娠16周～24周胎儿的羊水,采用G显带、G显带/FISH和G显带/FISH/CGH三阶梯的核型诊断程序,并分析其在产前诊断中的意义.结果 102例胎儿中,经第1阶梯诊断核型98例,诊断困难2例,失败2例;第2阶梯诊断核型2例,诊断困难1例,失败1例;第3阶梯诊断核型2例.经3阶梯诊断程序核型的诊断率达100%(102/102例),异常核型7例(7/102例,6.68 0A),其中第1、第2和第3阶梯分别诊断异常核型4例(4/7例,57.1 oA)、1例(1/7例,14.3%)和2例(2/7例,28.5%).结论 在产前诊断中实施3阶梯诊断程序有助于提高核型的确诊率,规范染色体诊断流程.     

Prenatal diagnosis of Charcot-Marie-Tooth disease type 1a by multicolor in situ hybridization

Genetic heterogeneity within the most common genetic neuropathy, Charcot-Marie-Tooth disease (CMT) result in about 70% slow nerve conduction CMT1 and 30% normal nerve conduction CMT2. Autosomal dominant CMT1A on chromosome 17p11.2 represents about 70% of CMT1 cases and about 50% of all CMT cases. Three different size CMT1A duplications with variable flanking breakpoints were characterized by multicolor in situ hybridization and confirmed by pulsed field gel electrophoresis and quantitative polymerase chain reaction (PCR) amplification. These different size duplications result in the same CMT1A phenotype confirming that trisomy of a normal gene region results in CMT1A. the smallest duplication does not include the 409 locus used previously to screen for CMT1A duplications. Direct analysis of interphase nuclei from fetuses and at-risk patients by multicolor in situ hybridization to a commonly duplicated CMT1A probe is informative more often than polymorphic PCR analysis, faster than pulsed field gel electrophoresis (PFGE), and faster, more informative, and more reliable than restriction enzyme analysis. CMT1B restriction enzyme analysis of CMT pedigrees without CMT1A is expected to diagnose another 8% of at-risk CMT1 patients (total: 78%). © 1993 Wiley-Liss, Inc.     

荧光原位杂交在恶性血液病中的研究进展

荧光原位杂交(fluorescence in situ hybtidization,FISH)可对分裂中期及间期的细胞进行染色体与基因异常检测,具有直观、快速、敏感性和特异性高以及方便灵活等优点,对白血病、淋巴瘤、多发性骨髓瘤、骨髓增生异常综合征等恶性血液病的诊断、分型、临床治疗选择和预后判断提供重要依据.     

荧光原位杂交在产前诊断Down′s综合征中的应用

目的探讨应用13/21α卫星探针荧光原位杂交(FISH)技术行产前诊断Down′s综合征(21-三体综合征)的价值。方法选择5例经细胞遗传学检查证实为孕正常胎儿孕妇的羊水细胞(对照组),5例经细胞遗传学检查证实为孕Down′s综合征胎儿孕妇的羊水细胞(观察组),用13/21α卫星探针对羊水细胞间期核进行荧光原位杂交。结果对照组和观察组总杂交率分别为34.6%和35.3%,无显著性差异(P>0.05)。两组含4个杂交信号的核平均百分比分别为30.1%和2.4%,含5个杂交信号的核平均百分比分别为2.2%和31.4%,有极显著性差异(P<0.01),。结论13/21α卫星探针间期FISH用于未培养的羊水细胞可以快速、准确地用于产前诊断Down综合征。     

Fiber-fluorescence in situ hybridization analyses as a diagnostic application for orientation of microduplications

Microduplications are normally invisible under microscopy and were not recognized before chromosomal microarray testing was available. Although it is difficult to confirm the orientation of duplicated segments by standard fluorescence in situ hybridization (FISH), our data indicates that fiber-FISH analysis has the potential to reveal the orientation of duplicated and triplicated segments of chromosomes. Recurrent microduplications reciprocal to microdeletions show tandem orientations of the duplicated segments, which is consistent with a non-allelic homologous recombination mechanism. Several random duplications showed tandem configurations and inverted duplications are rare. Further analysis is required to fully elucidate the basic mechanisms underlying such duplications/triplications.     

广西地区荧光原位杂交技术在快速诊断胎儿染色体数目异常中的应用

目的使用荧光原位杂交（FISH）技术对广西地区胎儿染色体数目异常进行快速检测并评估其临床应用价值。方法采用FISH技术对758例未培养羊水标本进行染色体数目异常的检测,采用染色体核型分析技术作为对照。结果成功检测758例标本,检出正常核型747例,异常核型11例（其中21-三体7例,18-三体1例,13-三体1例,XYY2例）,所有结果与染色体核型分析一致。结论FISH检测简便、快速、准确性高、特异性强,对协助临床咨询及产前遗传学诊断具有重要意义,值得推广应用。     

Characterization by fluorescence and electron microscopy in situ hybridization of a double Y isochromosome

A patient with mixed gonadal dysgenesis and Y isochromosomes i(Y) is described. Lymphocyte cultures from peripheral blood contained a high proportion of 45,X cells and several other cell lines with two different marker chromosomes (mars). These markers had either a monocentric (mar1) or a dicentric appearance (mar2). Following high-resolution GTG, RBG, QFQ, and CBG bandings, five cell lines were identified; 45,X/46,X, + mar1/46,X, + mar2/47,X, + mar1x2/47,X, + mar2x2. The percentages were 66/6/26/1/1%, respectively. Chromosome banding analyses were insufficient for characterization of the markers. In situ hybridization of specific probes for the Y centromere and its short arm showed, both in fluorescence and electron microscopy (EM), two different Y rearrangements. Mar1 is an isochromosome for the short arm i(Yp) and mar2 is a dicentric which was shown by EM to be a double isochromosome Yp, inv dup i(Yp). The breakpoint producing mar1 is within the centromere and the one producing mar2 is within one of the short arms of the Y isochromosome. The findings of different cell populations in peripheral blood lymphocytes indicate the postzygotic instability of this i(Yp). © 1995 Wiley-Liss, Inc.     

Prenatal diagnosis with repetitive in situ hybridization probes

We have used chromosome-specific repetitive sequences to detect the most common human aneuploidies prenatally. Together chromosome 21, 13, 18, X, and Y aneuploidy comprises 95% of the chromosome abnormalities that result in a high risk of abnormal phenotypes at birth. The X, Y, and 18 repetitive probes work reliably in multiple tissue types including directly examined and cultured amniocytes, chorionic villus cells, lymphocytes, and cultured fibroblasts. The probe that detects both chromosomes 13 and 21 routinely gives results in each cell type tested except directly studied amniocytes which can be interpreted in seven-ninths of the cases with protocol 1 and all tested samples with protocol 2. Our protocols diagnosed trisomy 21 in a 23-week fetus with low maternal serum AFP and a trisomy 18 in a direct chorionic villus sample 2 working days after the samples were obtained. Trisomy 21 also has been ruled out in a CVS karyotype first thought to be 47,XY,+21. These studies reflect the potential value of in situ hybridization to provide a more rapid, less expensive means to screen most at-risk fetal populations with less effort in first world cytogenetic laboratories, and to provide economical cytogenetic services in less developed countries. © 1992 Wiley-Liss, Inc.     

荧光原位杂交技术在诊断变异Ph易位及Ph(一)慢性髓细胞白血病中的应用

目的 探讨荧光原位杂交(fluorescence in situ hybridization,FISH)技术在诊断变异Ph易位及Ph(-)慢性髓细胞白血病(chronic myelocytic leukemia,CML)中的应用价值.方法 应用常规R显带方法,对9例伴有变异Ph易位和2例Ph(-)CML患者采用双色双融合bcr/abl探针进行FISH检测.结果 9例变异Ph易位CML患者异常核型除涉及9和22号染色体外,还涉及1、3、5、12、13、15、17、21号染色体,且部分类型为重现性异常,FISH结果均为阳性,信号特征为2R2G1Y;2例Ph(-)CML患者核型正常,FISH结果阳性,信号特征分别为1R1G2Y和1R1G1Y.结论 FISH技术对伴有变异Ph易位及Ph(-)CML患者的诊断更具有优势,可根据阳性细胞信号特征分析其异常核型,判断标记基因异常改变情况,是常规染色体显带分析的有益补充.     

荧光原位杂交技术检测多发性骨髓瘤复杂核型异常

目的探讨多重荧光原位杂交(multiplex fluorescence in situ hybridization,M-FISH)技术联合荧光原位杂交(fluorescence in situ hybridization,FISH)技术在检测多发性骨髓瘤(multiplemyeloma,MM)染色体异常中的应用价值及13q14缺失、IgH相关易位和17p13缺失的发生率。方法联合应用常规细胞遗传学(conventional cytogenetics,CC)方法及M-FISH和一组包括13q14(D13S319),14q32(IgH基因)和17p13(p53基因)探针的FISH技术分析了7例伴有复杂染色体异常的MM患者骨髓标本。结果M-FISH明确了CC分析中没有明确的异常,共检出12种染色体数目异常和29种结构异常,其中,1号染色体异常、13号染色体缺失和与14q32相关的易位最为多见。FISH检出6例伴有13q14缺失;4例伴有17p13缺失;5例伴有一个14q32相关易位,两例还伴有涉及两个14q32的易位。结论M-FISH联合FISH技术可以明确CC分析中复杂染色体异常,并发现和纠正CC分析中漏检及误检的异常,为MM染色体异常的研究提供了一种理想的     

Putative monosomy 21 in two patients: clinical findings and investigation using fluorescence in situ hybridization

Complete monosomy 21 is claimed to be a rare chromosomal disorder in which the cytogenetic investigation is bedevilled by technical difficulties. We describe the disparate clinical features in two patients in whom an initial diagnosis of monosomy 21 was made by routine karyotyping. Fluorescence in situ hybridisation (FISH) confirmed a translocation of chromosome 21 material to the short arm of chromosome 5 and to the X chromosome, respectively. The usefulness of FISH in the investigation of subtle chromosomal rearrangements is hereby demonstrated. These findings also cast doubt on the existence of "pure" monosomy 21 as an entity, and suggest that partial monosomy 21 is a more likely occurrence.     

Identification of mosaicism in Prader-Willi syndrome using fluorescent in situ hybridization

We report on our findings of 4 patients with mosaicism for a deletion of chromosome 15, most commonly associated with Prader-Willi syndrome (PWS). We examined a series of typical and atypical PWS patients in order to identify cytogenetically undetected deletions, using fluorescence in situ hybridization. In 4 of the patients analyzed we detected a deletion in 14–60% of peripheral blood leukocytes, using four commercially available probes. Our results indicate that mosaicism may play a role in the etiology of some PWS cases. These findings may be especially useful in patients who display discrepancies between clinical phenotype and established diagnostic criteria. Methylation and microsatellite polymorphism analyses of 2 patients with low-level mosaicism failed to identify the deletion. We propose that fluorescence in situ hybridization is the most effective method for detecting somatic mosaicism, since a large number of cells can be individually examined for the presence or absence of a specific deletion. © 1996 Wiley-Liss, Inc.