荧光原位杂交技术在染色体非整倍体产前诊断中的应用研究

目的建立荧光原位杂交技术应用于未培养羊水标本的染色体非整倍体产前诊断。方法对于符合产前诊断指征的孕妇,于孕16-25周抽取羊水25ml,其中20ml用于传统的细胞培养和染色体G显带分析,其余5ml用荧光原位杂交方法诊断13、8、21、X、Y染色体非整倍体。结果100例受检样本中,染色体数目异常3例,其中21三体2例,45,X 1例。FISH方法与传统羊水细胞核型分析符合率100%。结论FISH技术具有快速、简便、特异的特点,可作为部分染色体非整倍体的快速产前诊断方法。     

应用间期荧光原位杂交技术进行快速产前诊断

选用１８、Ｘ着丝粒探针及２１号染色体特异重复序列探针分别对未培养的羊水和绒毛细胞进行了间期荧光原位杂交。结果表明，常染色体探针出现３个及１个信号点的比例约为１％和６％。Ｘ染色体探针出现３点的比例也在１％左右，可为常染色体单体或三体以及Ｘ三体或多体提供诊断依据。同传统的细胞遗传学方法比较，间期荧光原位杂交具有简便、迅速、灵敏等优点，有一定临床应用价值     

应用优化荧光原位杂交技术诊断110例羊水间期细胞常见非整倍体

目的 优化现有荧光原位杂交(fluorescence in situ hybridization,FISH)技术,探讨FISH快速产前诊断多种染色体异常的临床应用.方法 改良FISH操作过程中的滴片和杂交液用量,对110例孕妇羊水样本同时进行FISH快速产前诊断和常规细胞培养核型分析.结果 110例羊水样本中,FISH检测出了4例21三体、1例18三体、58例46,XX、47例46,XY,与染色体核型分析的结果一致,符合率100%.结论 国产FISH试剂能快速、准确检测常见的5种染色体数日异常,使用样本量少、价廉.FISH技术可作为经典染色体核型分析的辅助方法,能应用于唐氏血清学高风险孕妇的快速产前诊断.     

Optimization of the fluorescence in situ hybridization (FISH) technique for high detection efficiency of very small proportions of target interphase nuclei

Using commercially available fluorochrome-labeled probes specific for chromosomes X, Y, 13, 18, and 21, we optimized the technical protocols for fluorescence in situ hybridization (FISH) so that the highest sensitivity and specificity were achieved. Also, we compared the optical properties of different types of fluorescent labels in an effort to develop the most efficient FISH protocol, including the determination of which types of labels are the easiest to count accurately. The lymphocytes were purified from blood of normal male and female newborns, normal male and female adults, and a trisomy 21 male adult. Male and female lymphocytes were mixed in five different combinations. For each combination, the male lymphocytes either from newborns or from adults were diluted with female lymphocytes in seven different proportions. For each of these 35 different cell mixtures, 100,000 nuclei were analyzed and scored in a blind fashion. Among the different fluorochrome-labeled probes, the highest sensitivity and specificity were achieved when SpectrumAqua CEP-Y/SpectrumOrange CEP X probe mixture, SpectrumAqua CEP-18, SpectrumOrange LSI-13, and SpectrumOrange LSI-21 were hybridized. The hybridization sensitivity and specificity were higher than 99% for the identification of chromosomes X, Y, 13, and 18, and higher than 98% for the detection of trisomy 21. The proportion of false-positive signals was under 0.005% for XY detection and lower than 0.14% for autosome detection. With these high hybridization sensitivities and specificities, the optimized FISH protocol developed in our laboratory has the potential to detect very rare events, e.g., when the proportion of cells being sought is lower than 0.01%. In other words, our protocol allows the specific detection of one male cell sunken among 10,000 female cells.     

Detection of aneuploidy in human spermatozoa using fluorescencein situ hybridization (FISH)

Summary Fluorescencein situ hybridization (FISH) with chromosome specific alpha-satellite DNA probes was used to estimate the rates of aneuploidy of chromosomes 1, 17, 18, X and Y in human ejaculated sperm. Sperm samples were collected from six donors, and biotinylated DNA probes, D1Z5, D17Z1, D18Z1, DXZ1 and DYZ3 were hybridized to interphase sperm which had been pretreated with dithiothreitol to expand their nuclei. A minimum of 3,000 sperm per donor were analyzed. The hybridization efficiency was 99.68% for all the five probes. The frequencies of aneuploidy for chromosomes 1, 17 and 18 were 0.65%, 0.66% and 0.61% respectively. For XX-and YY-sperm the frequencies were 0.28% and 0.27% respectively. To estimate the diploidy and disomy rates, a mixture of D17Z1 and D18Z1 were used as probes, and the frequency of diploid sperm was calculated to be 0.27%. After subtraction of the diploidy rate, the disomy rates for chromosomes 1, 17, and 18 were estimated to be 0.38%, 0.39% and 0.33%, respectively. The proportion of X- and Y-bearing sperm were 49.9% and 49.66%, consistent with an expected 1: 1 ratio.     

Use of fluorescence in situ hybridization for retrospective detection of aneuploidy in multiple myeloma

In malignancies with a low mitotic index such as multiple myeloma (MM), conventional cytogenetic studies may not be informative. This study's purpose was to assess specific numerical chromosomal aberrations in non-dividing MM cells by fluorescence in situ hybridization (FISH) of DNA chromosome probes on bone marrow smears. Old air-dried bone marrow smears from 18 MM patients were probed with alpha satellite DNA sequences for chromosomes 7, X, and Y, and a whole painting probe for chromosome 11. Plasma cells were identified by their morphologic characteristics so that counts of fluorescent signals in the nuclei of MM cells could be differentiated from those of normal marrow cells. Numerical chromosome aberrations were found in 66.7% of the cases (12 of 18), including 5 cases of trisomy 7, 2 cases of tetraploidy, 2 cases of monosomy X in females, 2 cases of disomy X in males, and 1 case of nullisomy Y. In addition, 2 of the 7 cases probed with chromosome 11 paint demonstrated 3 signals in about 15% of the cells. This study illustrates the advantages of FISH for interphase analysis of chromosome aberrations in slowly dividing cells, as well as the ability to use old slides for retrospective studies. © 1993 Wiley-Liss, Inc.     

Malignant cell detection by fluorescence in situ hybridization (FISH) in effusions from patients with carcinoma

Cytological diagnosis of malignant cells in effusions is hampered by difficulties in the differentiation from reactive mesothelial cells. Because interphase cytogenetics by fluorescence in situ hybridization (FISH) might complement cytological evaluation, we determined the power of tumor cell detection using FISH and cytology in 201 effusions from patients with advanced cancer. Furthermore, 9 primary breast tumors were FISH-karyotyped, and chromosomal aberrations were compared with those of corresponding metastatic effusion cells. By using centromeric probes representing chromosomes 7, 8, 11, 12, 17, and 18, a rate of malignancy-associated aneusomy combined for the 6 chromosomes was detected in an overall of 44.8% of effusion specimens (range, 31.8% to 39.3% for the individual chromosome), comparable to cytology (43.3%). The combination of just 2 FISH probes (namely, representing chromosome pairs 8/11 and 8/17) was almost equally efficient in the identification of aneusomy. Approximately one fourth of the cytologically negative effusions were FISH positive and vice versa. From the initially FISH-negative effusions, 18.9% could be subsequently classified positive with dual-color FISH by visualization of intranuclear chromosomal complexity in rare aneuploid cells. Thus, "overall FISH analysis," including dual-color evaluation, identified tumor cells in significantly more effusions (55.2%, P = .001) than conventional cytology, implying greater sensitivity. Finally, our finding that numerical aberration patterns in primary breast tumors and corresponding metastatic effusions are comparable indicates that FISH examination of primary tumors will indicate the centromeric probe(s) best suited for an efficient search for metastasis in the individual case.     

Position statement on interphase in situ hybridization prenatal diagnosis

Williams syndrome (WS) usually occurs sporadically. Few familial cases of Williams syndrome have been described, and those reports have often lacked photographic documentation. We describe 3 families, including a 3-year-old boy and his 34-year-old father, a 2-year-old girl and her 30-year-old mother, and a 3-year-old girl and her 31-year-old mother. None of these patients has supravalvular aortic stenosis or chromosome abnormalities. In all 3 families, the parent with Williams syndrome was diagnosed after the identification of the syndrome in the affected child. © 1993 Wiley-Liss, Inc.     

Specificity of interphase fluorescence in situ hybridization for detection of chromosome aberrations in tumor pathology

Interphase fluorescence in situ hybridization (IFISH) is an interesting and intriguing cytogenetic approach in the study of tumor chromosomal abnormalities when metaphases are not available. This technique can be applied to different types of tumor nuclei, including imprinted nuclei (IM), nuclei obtained from conventional cytogenetic procedures (PB), frozen nuclei, paraffin-embedded nuclei (PE), and nuclei extracted from paraffin-embedded sections (EX). IFISH is a high-sensitivity approach in tumor studies that can give evidence of genetic aberrations present in a small percentage of cells that are likely to escape detection if only molecular techniques are applied. Despite its high sensitivity and versatility, IFISH is an indirect cytogenetic method and needs controls to have adequate specificity. This study includes present data obtained in IFISH experiments using different types of probes (alpha-satellite and YAC clones) hybridized on different types of normal control nuclei, such as PB, IM, EX, and PE nuclei, to define the threshold level for monosomy and trisomy of different chromosomal regions. My findings demonstrate that the cut-off values depend both on the types of probes and on the types of target nuclei. Therefore, even if IFISH is a versatile, high-sensitivity technique for detecting chromosomal abnormalities, the lack of accurate controls may result in the misdiagnosis of some abnormalities.     

荧光原位杂交检测在产前诊断中的临床价值

目的探讨运用羊水间期细胞开展荧光原位杂交检测在产前诊断中的临床价值。方法运用FISH技术对110例孕16～24周孕妇的羊水间期细胞进行检测,每例均进行常规染色体核型分析。结果运用FISH法,所有样本均在19h内获得检测结果,除2例羊水培养失败外,所有样本均取得染色体核型分析报告结果。两种方法均检出特氏综合征、21-三体综合征各1例,所有样本的两种方法检测结果均互相符合。常规染色体核型分析有2例异常,因缺乏相应DNA探针。FISH法未能检出。结论运用羊水间期细胞开展荧光原位杂交检测,缩短了诊断时间,缓解了孕妇及家属因等待检测结果所产生的焦虑心情。检测程序简单,结果判定明确,在产前诊断实施过程中具有重要的临床价值。     

Cytologic and fluorescence in situ hybridization (FISH) examination of metanephric adenoma

Metanephric adenoma is a recently described benign renal neoplasm with distinctive histologic features. The cytologic appearance and fluorescence in situ hybridization (FISH) studies of this tumor have not been described. We present a case from a 48-yr-old woman. Cytologically, the cells were arranged in tight, short papillae and loose sheets. The cells had scant cytoplasm, round monotonous nuclei with fine even chromatin and rare small nucleoli. Immunohistochemistry revealed no reactivity for epithelial membrane antigen (EMA), keratins (AE1/AE3, callus, 34BE12), or carcinoembryonic antigen (CEA). FISH showed a disomic pattern for chromosomes 7, 17, and for the chromosome 3 short arm. The differential diagnosis includes Wilms' tumor, renal adenoma, papillary renal cell carcinoma, and metastatic tumors. Both immunohistochemistry and FISH may be of help in distinguishing some of these lesions. Diagn. Cytopathol. 16:107–111, 1997. © 1997 Wiley-Liss, Inc.     

用FISH技术快速诊断胎儿常见染色体数目异常

目的探讨荧光原位杂交(FISH)技术在产前诊断中的应用价值.方法采用13、18、21、X和Y染色体特异性DNA探针,对114例孕15～37w孕妇的羊水间期核进行FISH检测,同时行常规羊水细胞核型分析.结果与羊水细胞核型分析相符的染色体正常111例,异常3例,(47,XX 21、47,XY 21和46,XX,-21, t(21;21);另有1例核型为46,XY,t(15;18)(q26;q22),FISH信号显示正常.3例数目异常胎儿引产时抽脐血染色体检查结果与产前诊断一致.结论FISH技术用于产前诊断常见染色体数目异常,具有简便、快速、特异性强等优点,但有一定的局限性.应与常规核型分析相结合方可为临床提供更为真实可靠的信息.     

Rapid identification of beta-hemolytic streptococci by fluorescence in situ hybridization (FISH)

Rapid identification of pathogenic, beta-hemolytic streptococci is important for treatment decisions. We evaluated fluorescence in situ hybridization (FISH) for this purpose using 23 reference strains, 157 clinical isolates, and 80 blood cultures showing streptococci in the Gram stain. With a sensitivity and specificity in excess of 99%, FISH proved to be suitable for rapid identification of beta-hemolytic streptococci in a diagnostic laboratory.     

Mosaic dup (9p) diagnosed by fluorescence in situ hybridization (FISH)

We describe a girl with some manifestations of the dup (9p) syndrome. High-resolution Giemsa-banded karyotype of her lymphocytes documented that she was mosaic with 80% of cells being 46,XX, and 20% 46,XX,-20, + der(20;?) (p13;?). The additional material on 20p could not be defined clearly by high-resolution Giemsa banding, as the banding pattern appeared consistent with either distal 9p or distal 13q. In order to make a definitive cytogenetic diagnosis, we used fluorescence in situ hybridization (FISH) with a chromosome 9 specific DNA library to establish that the origin of the additional chromosomal material on chromosome 20 was from 9p. FISH used in this situation enabled us to counsel the family specifically regarding the prognosis and manifestations of distal 9p duplication. © 1993 Wiley-Liss, Inc.     

Prenatal detection of cri du chat syndrome on uncultured amniocytes using fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) with a chromosome-region-specific DNA probe was used prospectively on uncultured amniocyte interphase cells to detect an unbalanced chromosome abnormality that resulted in cri du chat or 5p – syndrome. Confirmation was performed by routine cytogenetics.     

Diagnosis of aneuploidy by in situ hybridization: Analysis of interphase nuclei

Moscow Research Institute of Pediatrics and Child Surgery, Ministry of Health of the RSFSR. All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR Yu. E. Vel'tishchev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 10, pp. 413–415, October, 1991.     

应用荧光原位杂交技术检测自然流产组织染色体数目异常

为评估荧光原位杂交技术在检测自然流产组织染色体数目异常中的价值和意义,本研究应用FISH技术检测105例自然流产绒毛或胎儿肌肉组织的13、21、18、X、Y染色体的数目,结果显示染色体数目异常的样本为45例,占其中42.86%;数目异常率大于60%的样本为39例;数目异常率介于10%-60%的样本为6例,其中早期流产组织中染色体异常的为29例,占64.44%;常见的数目异常类型为常染色体三体和X单体。与传统的制备染色体技术相比,FISH技术具有快速准确、稳定性高等特点,可以为查明流产原因提供依据,具有很高的临床应用价值。