共查询到20条相似文献,搜索用时 15 毫秒
1.
Pierre P. Massion Yong Zou Hasmet Uner Porntip Kiatsimkul Holly J. Wolf Anna E. Baron Tim Byers Steinn Jonsson Stephen Lam Fred R. Hirsch York E. Miller Wilbur A. Franklin Marileila Varella-Garcia 《PloS one》2009,4(6)
Lung carcinoma development is accompanied by field changes that may have diagnostic significance. We have previously shown the importance of chromosomal aneusomy in lung cancer progression. Here, we tested whether genomic gains in six specific loci, TP63 on 3q28, EGFR on 7p12, MYC on 8q24, 5p15.2, and centromeric regions for chromosomes 3 (CEP3) and 6 (CEP6), may provide further value in the prediction of lung cancer. Bronchial biopsy specimens were obtained by LIFE bronchoscopy from 70 subjects (27 with prevalent lung cancers and 43 individuals without lung cancer). Twenty six biopsies were read as moderate dysplasia, 21 as severe dysplasia and 23 as carcinoma in situ (CIS). Four-micron paraffin sections were submitted to a 4-target FISH assay (LAVysion, Abbott Molecular) and reprobed for TP63 and CEP 3 sequences. Spot counts were obtained in 30–50 nuclei per specimen for each probe. Increased gene copy number in 4 of the 6 probes was associated with increased risk of being diagnosed with lung cancer both in unadjusted analyses (odds ratio = 11, p<0.05) and adjusted for histology grade (odds ratio = 17, p<0.05). The most informative 4 probes were TP63, MYC, CEP3 and CEP6. The combination of these 4 probes offered a sensitivity of 82% for lung cancer and a specificity of 58%. These results indicate that specific cytogenetic alterations present in preinvasive lung lesions are closely associated with the diagnosis of lung cancer and may therefore have value in assessing lung cancer risk. 相似文献
2.
张宇曾庆山张宁熊廷川汪清 《现代生物医学进展》2012,12(7):1219-1222
目的:探讨荧光原位杂交技术辅助诊断膀胱尿路上皮癌的可行性。方法:标记为17号染色体着丝粒及9号染色体p16位点9p21区带探针,采用荧光原位杂交技术(Fluorescence in Situ Hybridization FISH)对80例膀胱肿瘤患者尿液间期细胞核进行荧光原位杂交,以20例健康志愿者作为正常对照组,建立阈值。以术后病理结果作为诊断"金标准",对80例膀胱肿瘤患者同时行尿脱落细胞学检查,与FISH进行比较。结果:17号染色体和9p21的畸变率分别为57.5%和63.8%。17号染色体畸变率主要表现为多倍体,与膀胱癌的分级有显著相关性(P<0.01);9号染色体畸变率主要变现为染色体缺失,与膀胱癌分期分级均无相关性(P>0.05)。尿脱落细胞学灵敏度为12.2%,FTSH技术灵敏度为86.5%;两者差异有统计学意义(P<0.01)。结论:荧光原位杂交技术可以作为膀胱尿路上皮癌诊断的一项重要方法,并可能在预后判断中具有重要临床意义。 相似文献
3.
Béla Kajtár Gábor Méhes Thomas L?rch Linda Deák Marika Kneifné Donát Alpár László Pajor 《Cytometry. Part A》2006,69(6):506-514
BACKGROUND: For chronic myeloid leukemia, the FISH detection of t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes is an alternative method to bone marrow karyotyping for monitoring treatment. With automation, several drawbacks of manual analysis may be circumvented. In this article, the capabilities of a commercially available automated image acquisition and analysis system were determined by detecting t(9;22)(q34;q11) in interphase nuclei of peripheral leukocytes. METHODS: Three peripheral blood samples of normal adults, 21 samples of CML patients, and one sample of a t(9;22)(q34;q11) positive cell-line were used. RESULTS: Single nuclei with correctly detected signals amounted to 99.6% of nuclei analyzed after exclusion of overlapping nuclei and nuclei with incorrect signal detection. A cut-off value of 0.84 mum was defined to discriminate between translocation positive and negative nuclei based on the shortest distance between signals. Using this value, the false positive rate of the automated analysis for negative samples was 7.0%, whereas that of the manual analysis was 5.8%. Automated and manual results showed strong correlation (R(2) = 0.985), the mean difference of results was only 3.7%. CONCLUSIONS: A reliable and objective automated analysis of large numbers of cells is possible, avoiding interobserver variability and producing statistically more accurate results than manual evaluation. 相似文献
4.
Kuang-Hua Chen Sheng-Chi Hsu Hou-Yu Chen Kwai-Fong Ng Tse-Ching Chen 《Biochemical and biophysical research communications》2014
Discrimination between complete moles (CMs), partial moles (PMs), and hydropic abortions (HAs) is important as the risk of persistent gestational trophoblastic disease (GTD) differs for each condition. We evaluated whether ancillary fluorescence in situ hybridization (FISH) with a set of chromosome enumeration probes (CEP) for chromosomes X, Y, and 17 and p57 immunostaining could improve the clinical diagnosis. Forty-one products of conception (POC) were reclassified according to clinical performance, morphology, p57 immunostaining results, and FISH results. The accuracy of histological examination alone was 85% for the original diagnosis. FISH analysis showed diploidy in 19 of 20 CMs and triploidy in 4 of 6 PMs. The concordance rate was 92.5% on using the CEP probes. p57 Staining was negative in all CMs and positive in all PMs and HAs. Chromosomal abnormality was detected in 3 cases of HA by using FISH. In conclusion, combined p57 immunostaining and FISH with a set of 3 CEP probes for chromosomes X, Y, and 17 could be useful in the classification of hydatidiform moles. 相似文献
5.
Chadwick B Willmore-Payne C Holden J Layfield L 《Analytical and quantitative cytology and histology / the International Academy of Cytology [and] American Society of Cytology》2007,29(6):351-357
OBJECTIVE: To investigate a series of tissues to determine if proliferation rate can affect chromosome counts by fluorescence in situ hybridization (FISH). STUDY DESIGN: We studied 9 non-neoplastic tissues and a trisomy 7 and tetrasomy 13 cell line by FISH. For each sample, 100 cells were analyzed for chromosome 7 and 13 number and MIB-1 expression. Centrometric enumeration probe (CEP) 7 counts were correlated with proliferation index. RESULTS: Average CEP 7 number showed a relationship to proliferation index, with higher CEP 7 averages associated with higher proliferation indices. Specimens of brain tissue demonstrated average CEP 7 counts between 1.64 and 1.75. Tissues with high proliferation indices (23-66%) demonstrated CEP 7 counts between 2.14 and 2.31. The average CEP 7 count for the trisomy 7 cell line was 2.61. The average LSI 13 count for the tetrasomy 13 cell line was 3.65. CONCLUSION: Chromosome 7 FISH counts demonstrated overlap between diploid tissues with high proliferation and triploid chromosome 7 tissues. This overlap was seen when 95% CI limits were used. The trisomic 7 and tetrasomic 13 cell lines demonstrated average CEP 7 and CEP 13 levels below 3 and 4, respectively. Definitions used for determination of polysomy should take into account tissue proliferation and section thicknesses. 相似文献
6.
7.
Ivan Y Iourov Ilia V Soloviev Svetlana G Vorsanova Viktor V Monakhov Yuri B Yurov 《The journal of histochemistry and cytochemistry》2005,53(3):401-408
A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies. 相似文献
8.
Hyperdiploidy and apparent aneusomy in mesothelial cells from non-malignant effusions as detected by fluorescence in situ hybridization (FISH) 总被引:1,自引:0,他引:1
Interphase cytogenetics by fluorescence in situ hybridization (FISH) can be used to detect malignant cells characterized by chromosomal aneuploidy. However, apparent aneusomy in normal "control" tissues has to be considered when using FISH as diagnostic tool. In effusions as model tissue exposed to metastasis, the definition of cut-off levels for background aneusomy by FISH was aimed in this study. Using centromeric probes representing chromosomes 7, 8, 11, 12, 17 and 18, extensive chromosome copy number enumeration by single-color FISH analysis was performed in pleural and ascitic effusions derived from 15 patients with various, non-malignant diseases. In all effusions, cells with gain of hybridization signals for several or all chromosomes tested were found (in up to 1.94% of cells). A consistent finding was high grade hyperdiploidy (>4 centromeric signals). Mesothelial elements mainly contributed to hyperdiploidy in effusions, as demonstrated by a combined analysis of FISH and immunocytochemistry with staining for cytokeratin. Dual-color FISH analysis showed that hyperdiploidy was predominantly corresponding to polyploidization; however, there were always minor cell populations classified as aneuploid by dual-color FISH. In conclusion, stringent criteria have to be applied to distinguish malignancy-related aneuploidy from background aneusomy by FISH. 相似文献
9.
Ashish Fauzdar Mohit Chowdhry R. N. Makroo Manoj Mishra Priyanka Srivastava Richa Tyagi Preeti Bhadauria Anita Kaul 《Indian journal of human genetics》2013,19(1):32-42
BACKGROUND AND OBJECTIVE:
Women with high-risk pregnancies are offered prenatal diagnosis through amniocentesis for cytogenetic analysis of fetal cells. The aim of this study was to evaluate the effectiveness of the rapid fluorescence in situ hybridization (FISH) technique for detecting numerical aberrations of chromosomes 13, 21, 18, X and Y in high-risk pregnancies in an Indian scenario.MATERIALS AND METHODS:
A total of 163 samples were received for a FISH and/or a full karyotype for prenatal diagnosis from high-risk pregnancies. In 116 samples both conventional culture techniques for getting karyotype through G-banding techniques were applied in conjunction to FISH test using the AneuVysion kit (Abbott Molecular, Inc.), following standard recommended protocol to compare the both the techniques in our setup.RESULTS:
Out of 116 patients, we got 96 normal for the five major chromosome abnormality and seven patients were found to be abnormal (04 trisomy 21, 02 monosomy X, and 01 trisomy 13) and all the FISH results correlated with conventional cytogenetics. To summarize the results of total 163 patients for the major chromosomal abnormalities analyzed by both/or cytogenetics and FISH there were 140 (86%) normal, 9 (6%) cases were abnormal and another 4 (2.5%) cases were suspicious mosaic and 10 (6%) cases of culture failure. The diagnostic detection rate with FISH in 116 patients was 97.5%. There were no false-positive and false-negative autosomal or sex chromosomal results, within our established criteria for reporting FISH signals.CONCLUSION:
Rapid FISH is a reliable and prompt method for detecting numerical chromosomal aberrations and has now been implemented as a routine diagnostic procedure for detection of fetal aneuploidy in India. 相似文献10.
Rossi E Ubiali A Balzarini P Cadei M Alpi F Grigolatoi PG 《European journal of histochemistry : EJH》2005,49(1):53-58
A novel application of fluorescence in situ hybridization (FISH) to isolated nuclei is described. The method detects gene amplification and chromosome aneuploidy in extracted nuclei from paraffin-embedded tissue of human cancer with greater sensitivity and specificity than existing FISH methods. In this study, the method is applied to signal detection of the HER-2/neu (c-erbB-2) gene, whose amplification is one of the most common genetic alterations associated with human breast cancer. Nuclei were extracted and isolated from formalin fixed, paraffin embedded tissue of 43 different carcinomas (breast, ovary, endometrium, gastrointestinal stromal tumor and malignant mesothelioma). FISH was performed both on sections and extracted nuclei of each tissue using chromosome enumeration probes (CEP) for the centromeric regions of chromosomes 8 and 17, and a locus specific identifier (LSI) for the HER-2/neu oncogene. Differences between ploidy calculated in sections and extracted nuclei were seen in 3 breast carcinomas and 1 gastrointestinal stromal tumor (GIST). Furthermore, 1 breast cancer, previously considered to be borderline for HER-2/neu gene amplification turned out to be clearly amplified. Nuclei extraction and isolation bypass all the problems related to signal interpretation in tissue sections, and the adoption of this new technique, which improves the signal quality in several neoplastic samples, is suggested. 相似文献
11.
Background
Human epidermal growth factor receptor 2 (HER2) fluorescence in situ hybridization (FISH) is a quantitative assay for selecting breast cancer patients for trastuzumab therapy. However, current HER2 FISH procedures are labor intensive, manual methods that require skilled technologists and specialized fluorescence microscopy. Furthermore, FISH slides cannot be archived for long term storage and review. Our objective was to develop an automated brightfield double in situ hybridization (BDISH) application for HER2 gene and chromosome 17 centromere (CEN 17) and test the assay performance with dual color HER2 FISH evaluated breast carcinomas.Methods
The BDISH assay was developed with the nick translated dinitrophenyl (DNP)-labeled HER2 DNA probe and DNP-labeled CEN 17 oligoprobe on the Ventana BenchMark® XT slide processing system. Detection of HER2 and CEN 17 signals was accomplished with the silver acetate, hydroquinone, and H2O2 reaction with horseradish peroxidase (HRP) and the fast red and naphthol phosphate reaction with alkaline phosphatise (AP), respectively. The BDISH specificity was optimized with formalin-fixed, paraffin-embedded xenograft tumors, MCF7 (non-amplified HER2 gene) and BT-474 (amplified HER2 gene). Then, the BDISH performance was evaluated with 94 routinely processed breast cancer tissues. Interpretation of HER2 and CEN 17 BDISH slides was conducted by 4 observers using a conventional brightfield microscope without oil immersion objectives.Results
Sequential hybridization and signal detection for HER2 and CEN 17 ISH demonstrated both DNA targets in the same cells. HER2 signals were visualized as discrete black metallic silver dots while CEN 17 signals were detected as slightly larger red dots. Our study demonstrated a high consensus concordance between HER2 FISH and BDISH results of clinical breast carcinoma cases based on the historical scoring method (98.9%, Simple Kappa = 0.9736, 95% CI = 0.9222 – 1.0000) and the ASCO/CAP scoring method with the FISH equivocal cases (95.7%, Simple Kappa = 0.8993%, 95% CI = 0.8068 – 0.9919) and without the FISH equivocal cases (100%, Simple Kappa = 1.0000%, 95% CI = 1.0000 – 1.0000).Conclusion
Automated BDISH applications for HER2 and CEN 17 targets were successfully developed and it might be able to replace manual two-color HER2 FISH methods. The application also has the potential to be used for other gene targets. The use of BDISH technology allows the simultaneous analyses of two DNA targets within the context of tissue morphological observation. 相似文献12.
We describe the application of lamin immunocytochemistry (ICC) and single- or double-target fluorescence in situ hybridization (FISH) on 4 microm thick frozen tissue sections as a method to facilitate scoring of aberrant chromosome copy numbers in colonic tumors. Analysis of FISH signals in colon tissue sections is often hampered by overlap and truncation of epithelial nuclei, due to the density of the epithelial cells. Furthermore, on the basis of nuclear staining it is often difficult to determine whether or not nuclei are overlapping, or adjoining. Therefore, reliable evaluation of (F)ISH signals to screen for genomic changes was until now mainly restricted to isolated nuclei obtained from relatively thick tissue sections. In this study the applicability of lamin ICC, to stain the nuclear periphery and to distinguish individual nuclei, combined with the FISH procedure is explored to solve this problem for colon epithelium. For ICC we applied the alkaline phosphatase (APase)-Fast Red detection method, since the fluorescent precipitate of this reaction resists extensive proteolytic digestion as needed for efficient FISH on tissue sections. Chromosome copy numbers could easily be determined in 4 microm thick frozen tissue sections by combining lamin ICC and FISH. The ratio of the copy numbers of the chromosomes 7 and 17 could be determined in frozen tissue sections after combined lamin ICC and double-target FISH. It is concluded that the combination of lamin ICC and FISH improves chromosome copy number analysis and can be used to investigate genomic changes in different tumor compartments in thin frozen tissue sections. 相似文献
13.
Rita Narath Thomas L?rch Karin M Greulich-Bode Petra Boukamp Peter F Ambros 《Cytometry. Part A》2005,68(2):113-120
To benefit from the fluorescence-based automatic microscope (FLAME), we have adapted a PNA FISH technique to automatically determine telomere length in interphase nuclei. The method relies on the simultaneous acquisition of pan-telomeric signals and reference probe signals. We compared the quantitative figures to those for existing methods, i.e. Southern blot analysis and quantitative FISH (Q-FISH). Quantitative-FISH on interphase nuclei (IQ-FISH) allows the exact quantification of telomere length in interphase nuclei. Thus, this enables us to obtain not only exact information on the telomere length, but also morphological and topological details. The automatic measurement of large cell numbers allows the measurement of statistically relevant cell populations. 相似文献
14.
In the present study, we developed a fluorescence in situ hybridization (FISH) strategy, which allows a reliable determination of the chromatid number of specific chromosomes in mature human oocytes. 168 unfertilized oocytes were analyzed by dual-color FISH with two direct-labeled locus-specific DNA probes for chromosome 13 and 21. To exclude FISH failures, metaphases with abnormal signal patterns were reanalyzed by multi-locus-FISH (ML-FISH) for chromosome 13 and 21. Following dual-color FISH, abnormal signal patterns were detected in 21 out of 108 metaphases (19.4%). 17 of these metaphases were reanalyzed by ML-FISH. In contrast to the first FISH, seven metaphases showed normal signal patterns after rehybridization, whereas ten metaphases remained abnormal. Out of these real aneuploid metaphases, five showed gain or loss of a single signal (= chromatid), two showed missing double signals (= chromosome) and three showed both. In conclusion, locus-specific FISH probes facilitate differentiation between first meiotic nondisjunction of whole chromosomes and prematurely divided chromatids. Moreover, simultaneous hybridization with a second locus-specific probe on the same chromatid (ML-FISH) helps to differentiate between FISH failures and real meiotic division errors and therefore, allows a more reliable analysis of aneuploidies in human oocytes. 相似文献
15.
Bink K Walch A Feuchtinger A Eisenmann H Hutzler P Höfler H Werner M 《Histochemistry and cell biology》2001,115(4):293-299
Confocal laser scanning microscopy (CLSM) is an extensive but reliable tool for assessing the hybridisation signals in fluorescence in situ hybridisation (FISH). Most CLSMs are equipped with an argon-laser and a helium/neon-laser illumination system with excitation wavelengths of 488, 543 and 633 nm. A protocol for an optimal nuclear counterstaining in combination with dual-colour FISH for these laser illumination systems has not been established so far. Here, we determined the suitability of eleven dimeric and monomeric cyanine nucleic acid stains on paraffin sections of breast carcinoma specimens in combination with dual-colour FISH (Her-2/neu and centromere 17) for CLSM application. Strong staining of cell nuclei was observed for TO-PRO-3 and YO-PRO-3, YOYO-1 and propidium iodide (PI), but only TO-PRO-3 showed specific staining of nuclei without any staining of the cytoplasm. A specific emission in exclusively one distinct fluorescence channel was shown for TO-PRO-3 (633 nm excitation) as well as YOYO-1, BO-PRO-1 and Sytox Green (488 nm excitation), evaluated by a CLSM and confirmed by 3-D fluorescence spectra. High stability of fluorescence intensity was shown for the far-red dyes TO-PRO-3, YO-PRO-3, YOYO-3 and Syto-59 as well as YOYO-1 and PI. Only TO-PRO-3 was due to its high specificity and stability suitable for detection of an amplification of the Her-2/neu gene by dual-colour FISH and CLSM evaluation. 相似文献
16.
Amare PK Baisane C Nair R Menon H Banavali S Kabre S Gujral S Subramaniam P 《Indian journal of human genetics》2011,17(2):54-58
Acute promyelocytic leukemia (APL) is characterized by a reciprocal translocation t(15;17)(q22;q21) leading to the disruption of Promyelocytic leukemia (PML) and Retionic Acid Receptor Alpha (RARA) followed by reciprocal PML-RARA fusion in 90% of the cases. Fluorescence in situ hybridization (FISH) has overcome the hurdles of unavailability of abnormal and/or lack of metaphase cells, and detection of cryptic, submicroscopic rearrangements. In the present study, besides diagnostic approach we sought to analyze these cases for identification and characterization of cryptic rearrangements, deletion variants and unknown RARA translocation variants by application of D-FISH and RARA break-apart probe strategy on interphase and metaphase cells in a large series of 200 cases of APL. Forty cases (20%) had atypical PML-RARA and/or RARA variants. D-FISH with PML/RARA probe helped identification of RARA insertion to PML. By application of D-FISH on metaphase cells, we documented that translocation of 15 to 17 leads to 17q deletion which results in loss of reciprocal fusion and/or residual RARA on der(17). Among the complex variants of t(15;17), PML-RARA fusion followed by residual RARA insertion closed to PML-RARA on der(15) was unique and unusual. FISH with break-apart RARA probe on metaphase cells was found to be a very efficient strategy to detect unknown RARA variant translocations like t(11;17)(q23;q21), t(11;17)(q13;q21) and t(2;17)(p21;q21). These findings proved that D-FISH and break-apart probe strategy has potential to detect primary as well as secondary additional aberrations of PML, RARA and other additional loci. The long-term clinical follow-up is essential to evaluate the clinical importance of these findings. 相似文献
17.
应用双色荧光原位杂交技术检测克氏综合征 总被引:2,自引:1,他引:2
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. 相似文献
18.
Elise M. J. van der Logt Deborah A. J. Kuperus Jan W. van Setten Marius C. van den Heuvel James. E. Boers Ed Schuuring Robby E. Kibbelaar 《PloS one》2015,10(4)
HER2 assessment is routinely used to select patients with invasive breast cancer that might benefit from HER2-targeted therapy. The aim of this study was to validate a fully automated in situ hybridization (ISH) procedure that combines the automated Leica HER2 fluorescent ISH system for Bond with supervised automated analysis with the Visia imaging D-Sight digital imaging platform. HER2 assessment was performed on 328 formalin-fixed/paraffin-embedded invasive breast cancer tumors on tissue microarrays (TMA) and 100 (50 selected IHC 2+ and 50 random IHC scores) full-sized slides of resections/biopsies obtained for diagnostic purposes previously. For digital analysis slides were pre-screened at 20x and 100x magnification for all fluorescent signals and supervised-automated scoring was performed on at least two pictures (in total at least 20 nuclei were counted) with the D-Sight HER2 FISH analysis module by two observers independently. Results were compared to data obtained previously with the manual Abbott FISH test. The overall agreement with Abbott FISH data among TMA samples and 50 selected IHC 2+ cases was 98.8% (κ = 0.94) and 93.8% (κ = 0.88), respectively. The results of 50 additionally tested unselected IHC cases were concordant with previously obtained IHC and/or FISH data. The combination of the Leica FISH system with the D-Sight digital imaging platform is a feasible method for HER2 assessment in routine clinical practice for patients with invasive breast cancer. 相似文献
19.
In the case of chromosomal aneuploidy in sperm wherein the incident rate is low and a large number of cells require scoring, automated methods that rely on computer software to segment and to count fluorescence signals are particularly necessary due to countless hours spent in reading slides and to the potential for interoperator differences. The purpose of this pilot experiment was to determine whether there were significant differences in the estimates of disomy frequency produced by automated versus manual scoring of signals for chromosome X, Y, and 18 in human sperm. The frequency of X18, Y18, XX18, YY18, and XY18 were determined in four separate normozoospermic samples. Slides were hybridized using a standard sperm FISH protocol for centromere-specific probes. Between 500 and 564, DAPI positive nuclei were captured from each sample and scored using the automated system, and the same slides were scored by a trained cytogeneticist, who was blind to the purpose of the study and the automated system results. None of the estimated frequencies was significantly different between manual and automated methods, regardless of whether individual slides or pooled results across all samples were compared. To our knowledge, this is the first report examining the validity of automated cell scoring in human spermatozoa. The results from this pilot exploration of sperm FISH suggest the comparability between automated and manual methods for estimating sex chromosome disomy and provide evidence that automated laser scanning of multiprobe sperm FISH should be explored further. 相似文献
20.
Heike Horn Julia Bausinger Annette M. Staiger Maximilian Sohn Christopher Schmelter Kim Gruber Claudia Kalla M. Michaela Ott Andreas Rosenwald German Ott 《PloS one》2014,9(4)
Few data are available regarding the reliability of fluorescence in-situ hybridization (FISH), especially for chromosomal deletions, in high-throughput settings using tissue microarrays (TMAs). We performed a comprehensive FISH study for the detection of chromosomal translocations and deletions in formalin-fixed and paraffin-embedded (FFPE) tumor specimens arranged in TMA format. We analyzed 46 B-cell lymphoma (B-NHL) specimens with known karyotypes for translocations of IGH-, BCL2-, BCL6- and MYC-genes. Locus-specific DNA probes were used for the detection of deletions in chromosome bands 6q21 and 9p21 in 62 follicular lymphomas (FL) and six malignant mesothelioma (MM) samples, respectively. To test for aberrant signals generated by truncation of nuclei following sectioning of FFPE tissue samples, cell line dilutions with 9p21-deletions were embedded into paraffin blocks. The overall TMA hybridization efficiency was 94%. FISH results regarding translocations matched karyotyping data in 93%. As for chromosomal deletions, sectioning artefacts occurred in 17% to 25% of cells, suggesting that the proportion of cells showing deletions should exceed 25% to be reliably detectable. In conclusion, FISH represents a robust tool for the detection of structural as well as numerical aberrations in FFPE tissue samples in a TMA-based high-throughput setting, when rigorous cut-off values and appropriate controls are maintained, and, of note, was superior to quantitative PCR approaches. 相似文献