Fluorescence in situ hybridization (FISH) for rapid detection of aneuploidy: experience in 911 prenatal cases†

Fluorescence in situ hybridization (FISH) was performed with probes specific for chromosomes 13, 18, 21, X and Y on 911 of 11123 (8.2%) amniotic fluid samples submitted to the present authors' laboratory for cytogenetic analysis over an 8-year period. Altogether 3516 hybridizations were performed with an interpretable FISH result on all chromosomes requested in 884/911 (97%) of cases. An uninformative FISH result occurred in 44 hybridizations among 27 cases (3%). Of a total of 89 karyotypically proven cases with aneuploidy that might have been detected by FISH, the overall detection rate was 84%. An inconclusive or incomplete FISH result occurred in 9/89 (10%) of these proven aneuploid cases. In the remaining 80 informative proven aneuploid cases, correct detection of aneuploidy was accomplished in 75/80 (94%) of samples. A false-negative result occurred in the remaining 5/80 (6%) of such informative cases. Eighteen cases had karyotypically proven abnormalities that could not have been detected by the targeted FISH. Aside from these 18 cases, FISH allowed correct detection of normal disomy in 785/804 (98%) of such cases. An incomplete FISH result occurred in 18 normal disomic cases. There was a single possible ‘false-positive’ FISH result for chromosome 21. Interphase FISH analysis of uncultured amniotic fluid cells has been shown to be a useful laboratory tool for rapid fetal aneuploidy screening during pregnancy. As with all clinical laboratory diagnostic tests, incomplete or inconclusive results (or even interpretive errors) occur in a small percentage of cases. Nevertheless, FISH results accompanied by other data and by appropriate counseling provide clinicians and patients with valuable information for clinical decision-making surrounding family planning and pregnancy management. Copyright © 2001 John Wiley & Sons, Ltd.     

Preimplantation genetic diagnosis using FISH for carriers of Robertsonian translocations: the Portuguese experience

Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis for couples at risk of transmitting genetic disorders to their offspring. We present a fluorescence in situ hybridization (FISH) analysis of embryos obtained after seven PGD cycles in six couples with Robertsonian translocations and male factor infertility: 4 der(13;14), 1 der(14;21) and 1 der(15;21). Of 74 metaphase II (MII) injected oocytes, 61 (82.4%) fertilized normally and cleaved. Of these, 37/61 (60.7%) embryos were of high morphological quality with ≥6 blastomeres. After biopsy of 44 embryos at day 3 of development, seven degenerated, seven arrested in development and 30/44 (68.2%) evolved, of which 25/30 (83.3%) reached the morula/blastocyst stage. Analysis of biopsied blastomeres showed 23/44 (52.3%) of normal/balanced embryos, of which 15 (11 at the morula/blastocyst stage) were transferred in six cycles. One term pregnancy was achieved, which ended by cesarean section at 37 weeks of gestation, giving birth to two healthy newborn. Analysis of 49 embryos (excluding 12 inconclusive cases) showed a predominance of alternate segregation (38/49, 77.6%) over adjacent segregation (7/49, 14.3%), with one (2%) being a polyploid mosaic and three (6.1%) chaotic. Copyright © 2002 John Wiley & Sons, Ltd.     

应用荧光原位杂交方法检测中国沿海塔玛/链状亚历山大藻复合种(亚洲温带基因型)

近年来,有害赤潮于中国沿海频繁发生,对沿海居民身体健康、水产养殖和自然生态形成了潜在的威胁.由于部分有毒赤潮藻在很低的密度下就有可能导致严重的危害,但传统的采样和分析方法无法对这类有毒赤潮进行有效的检测,因而急需发展准确、高效的检测新方法.根据对中国沿海分离的塔玛/链状亚历山大藻(亚洲温带基因型)核糖体大亚基DNA(LSU rDNA)序列信息的分析,设计了两条特异性的荧光标记探针,并建立了针对中国沿海塔玛/链状亚历山大藻复合种(亚洲温带基因型)的荧光原位杂交检测方法室内模拟实验显示两条探针都能够特异性地标记中国沿海塔玛/链状亚历山大藻(亚洲温带基因型),但标记效果有一定差异,探针SPEC-PROBE2标记效果远好于探针SPEC-PROBE1.经过标记的藻细胞可以通过荧光显微镜和流式细胞仪区分.中国沿海塔玛/链状亚历山大藻荧光原位杂交检测方法的建立将有助于提高海水样品中亚历山大藻监测的准确性和工作效率.     

Role of amniotic fluid interphase fluorescence in situ hybridization (FISH) analysis in patient management

We retrospectively reviewed 309 amniotic fluid interphase fluorescence in situ hybridization (FISH) analyses performed from October 1995 to June 1999 to assess the role of interphase FISH in the management of patients at increased risk for fetal aneuploidies. Gestational age and indications for amniocentesis, clinical interventions after FISH results, as well as interventions after final culture reports were analyzed. There were 244 (79%) normal, 50 (16%) abnormal and 15 (5%) inconclusive FISH results. There were no false-positive or false-negative results, but there were nine (3%) clinically significant chromosomal abnormalities not detectable by FISH. Of the 50 women with abnormal FISH results, 26 (52%) elected to terminate the pregnancy prior to the availability of the standard chromosome analysis. In two of the fetuses with trisomy 21 no abnormalities were reported by ultrasound examination. Our experience indicates that interphase FISH results played an important role in decision making, especially for pregnancies close to 24 weeks' gestation. Standard karyotype analysis is still required for detection of chromosome abnormalities not detectable by interphase FISH techniques and for clarification of unusual or inconclusive FISH results. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal aneuploidy detection in interphase cells by fluorescence in situ hybridization (Fish)

FISH is a quick, inexpensive, accurate, sensitive and relatively specific method for aneuploidy detection in samples of uncultured chorionic villus cells and amniotic fluid cells. FISH allows detection of the autosomal trisomies 13, 18 and 21 and X and Y abnormalities and any other chromosome abnormality for which a specific probe is available. The detection rate of these abnormalities is high in informative samples which have a concordance of > 99.5% with cytogenetic results. A relatively high number of abnormal cases are found in uninformative samples. However, such samples should be regarded as samples to be investigated further. Clinical experience with the use of FISH for prenatal diagnosis is now beyond 10,000 cases; a number of clinical protocols and smaller trials have also been carried out, resulting in 90% of attempted analyses giving informative results with a high detection rate and extraordinarily low false-positive and false-negative rates Unsolved problems remain, such as occasional technical failures, admixtures of maternal blood and up to 20% uninformative scoring results, especially for abnormal specimens. FISH is at present used as an adjunct to classical cytogenetic analysis. However, this should not be interpreted as meaning that FISH could not be used as a methodology in its own right. If FISH were to be considered a Diagnostic test then this might be the case, due to the risk of false-negative and false-positive results and the fact that FISH does not allow a diagnosis of certain structural abnormalities. If, on the other hand, FISH is considered a screening test, which means that in all abnormal (or indeterminate) cases, classical cytogenetic analysis would follow the abnormal screening test, the accuracy which is potentially higher than for other screening methods, for example in cases of trisomy 21, justifies FISH as a prenatal screening test in its own right.     

Magnetic-activated cell sorting (MACS) significantly decreases the hybridization efficiency of fluorescence in situ hybridization (FISH)

Fetal cells were enriched from maternal blood using density gradient centrifugation of Histopaque followed by magnetic-activated cell sorting (MACS) to select CD71-positive cells. For each specimen, cells partially purified by Histopaque were split into equal portions, and each portion was subjected to purification by MACS in parallel. Cells before and after MACS were subjected to dual-color fluorescence in situ hybridization (FISH) analysis with X- and Y-chromosome-specific probes. We found that the hybridization rates were decreased by approximately 10% after MACS based on duplicated analysis for each sample. Copyright © 2001 John Wiley & Sons, Ltd.     

Distribution characteristics of ammonia-oxidizing bacteria in the Typha latifolia constructed wetlands using fluorescent in situ hybridization(FISH)

A molecular biology method, fluorescent in situ hybridization（FISH）, in which the pre-treatment was improved in allusion to the media of the constructed wetlands（CW）, e.g. the soil and the grit, was used to investigate the vertical distribution characteristics of ammonia-oxidizing bacteria（AOB） quantity and the relation with oxidation-reduction potential（ORP） in the Typha latifolia constructed wetlands under three different Ioadings in summer from May to September. Results showed that the quantity of the AOB decreased in the Typha latifolia CW with the increase of vertical depth. However, the AOB quantity was 2-4 times the quantity of the control in the root area. Additionally, ORP in the rhizosphere was found to be higher than other areas, which showed that Typha latifolia CW was in an aerobic state in summer when using simulated non-point sewage at the rural area of Taihu Lake in China and small town combined sewage.     

Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH): 2-year multi-center retrospective study and review of the literature

Since 1993, the position of the American College of Medical Genetics (ACMG) has been that prenatal interphase fluorescence in situ hybridization (FISH) is investigational. In 1997, the FDA cleared the AneuVysion^® assay (Vysis, Inc.) to enumerate chromosomes 13, 18, 21, X and Y for prenatal diagnosis. Data is presented from the clinical trial that led to regulatory clearance (1379 pregnancies) and from retrospective case review on 5197 new pregnancies. These studies demonstrated an extremely high concordance rate between FISH and standard cytogenetics (99.8%) for specific abnormalities that the AneuVysion assay is designed to detect. In 29 039 informative testing events (6576 new and 22 463 cases in the literature) only one false positive (false positive rate=0.003%) and seven false negative results (false negative rate=0.024%) occurred. A historical review of all known accounts of specimens tested is presented (29 039 using AneuVysion and 18 275 specimens tested with other probes). These performance characteristics support a prenatal management strategy that includes utilization of FISH for prenatal testing when a diagnosis of aneuploidy of chromosome 13, 18, 21, X or Y is highly suspected by virtue of maternal age, positive maternal serum biochemical screening or abnormal ultrasound findings. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal detection of trisomy 21 in uncultured amniocytes by fluorescence in situ hybridization: A prospective study

A prospective study was undertaken to evaluate the use of fluorescence in situ hybridization (FISH) for the detection of trisomy 21 in interphase nuclei of uncultured amniotic fluid cells. Five hundred cases were analysed in situ and classified as normal or abnormal; the results were subsequently checked against the cytogenetic findings. Four hundred and ninety-three were correctly identified as normal with an 86·6 per cent average frequency of scored nuclei exhibiting two signals; six cases were correctly identified as trisomic for chromosome 21 with 81·7 per cent of scored nuclei exhibiting three signals; and one abnormal case involving an unbalanced chromosome 21·21 translocation was falsely scored as normal due to poor hybridization/detection efficiency. The method has been substantially improved and simplified so that it is suitable for the rapid detection of trisomy 21. As aneuploidy detection in interphase does not identify structural chromosome aberrations, it is not a substitute for fetal chromosome analysis.     

Prenatal application of fluorescent in situ hybridization (FISH) for identification of a mosaic Y-chromosome marker,IDIC(Yp)

An amniocentesis was performed at 13.3 weeks' gestation for advanced maternal age. A mosaic sex chromosome pattern was found: of 50 cells examined, 34 had a 45,X karyotype. In 14 cells with a modal number of 46, a recognizable Y was substituted by a small non-fluorescent marker. C-banding identified the marker as an isodicentric in 12 cells. In two cells, the non-fluorescent marker appeared to be monocentric and looked like a non-fluorescent del (Yq), but could have been an isodicentric Y with inactivation of one of the centromeres. Two cells with a modal number of 47 showed two copies of the monocentric marker. Fluorescent in situ hybridization with an alpha satellite Y-specific centromeric probe confirmed the Y-chromosome origin of the markers and allowed for more accurate prenatal diagnostic information.     

FISH法作为厌氧消化系统运行管理指标的可行性

以高浓度厌氧消化污泥为对象,进行FISH、MPN和产甲烷活性的测定实验.FISH分析得到,厌氧消化污泥中古细菌、嗜乙酸产甲烷菌和嗜氢产甲烷菌占全菌数的比例分别为30%～70%,18%～28%和15%～43%,菌数测定结果FISH法高于MPN法.研究表明,FISH法可用于高浓度厌氧消化污泥的菌相解析,作为处理系统运行管理指标使用.     

Prenatal identification of a 45,X/46,Xder(Y) mosaicism and confirmation by high resolution cytogenetics and fluorescence in situ hybridization

A 45,X/46,Xder(Y) mosaicism detected prenatally was shown to have a rare Y inversion- duplication or Y/Y translocation which can only be identified by a combination of high resolution cytogenetics and fluorescence in situ hybridization. The present data indicate the usefulness and importance of chromosome-specific probes in the identification and characterization of chromosome rearrangements.     

Prenatal diagnosis of the derivative chromosome 22 associated with cat eye syndrome by fluorescence in situ hybridization

Cytogenetic studies of cultured amniocytes demonstrated a karyotype of 46, XX/47, XX,+mar. A bisatellited, dicentric, distamycin-DAPI negative, NOR-positive marker was present in 76 per cent of the metaphases examined. Similar markers have been associated with cat eye syndrome (CES). We report on the utilization of fluorescence in situ hybridization (FISH) with a 14/22 a-satellite probe and a chromosome 22-specific cosmid for locus D22S9 to determine the origin of the prenatally detected supernumerary marker chromosome. FISH studies demonstrated that the marker is a derivative of chromosome 22 and enabled us to provide the family with additional prognostic information.     

Fluorescent in situ hybridization (FISH): A new application in the delineation of true vs. pseudomosaicism in prenatal diagnosis

Metaphase chromosomes and interphase nuclei from nine amniotic fluid cultures were studied with fluorescence in situ hybridization (FISH). The samples were initially analyzed with routine G-banding and were diagnosed as having true mosaicism (five patients) or pseudomosaicism (four patients). In our study, FISH analysis could provide additional information to distinguish pseudo– from true mosaicism by allowing interphase studies and analysis of an increased number of metaphase spreads. These results suggest a multilinear origin of ‘in situ’ colonies of cells.     

Preimplantation genetic diagnosis (PGD), a collaborative activity of clinical genetic departments and IVF centres

An Erratum has been published for this article in Prenatal Diagnosis 22 (5) 2002, 451. Preimplantation genetic diagnosis (PGD) requires the combined efforts of geneticists and workers in the field of reproductive medicine. This was studied on the basis of a questionnaire, sent to 35 members of the PGD Consortium of the European Society of Human Reproduction and Embryology (ESHRE). A reply was obtained from 20 centres. They represent the majority of activities in the field of PGD in the world. It is obvious that many of the activities (in vitro fertilisation, embryo culture and biopsy) take place in IVF units while others (counselling and diagnosis) are the responsibility of genetic diagnostic centres. The distances between both units vary considerably. In all but one centre sex determination is offered. Aneuploidy screening is offered in 13 out of 20 centres. PGD of translocations and other structural chromosome abnormalities is offered in all but one centre. The number of monogenic diseases offered varies considerably. In comparison to prenatal diagnosis PGD is more expensive. The majority of these costs are due to the IVF or ICSI procedure. The charges for PGD vary between about € 600 and € 4000. In 16 out of 20 centres the parents to be must sign an informed consent form. Copyright © 2001 John Wiley & Sons, Ltd.