应用荧光原位杂交技术诊断羊水细胞染色体异常

探讨荧光源位杂交技术于产前诊断羊水细胞染色体异常的实验方法的应用价值。方法对３４例孕１６－２３周有产前认旨征者经腹部抽取羊水,用Ｃｈａｎｇ培养液传代培养,常规制备羊水细胞分裂中期染色体,应用生物素及地高辛标记的人类全着线粒探针和Ｘ、Ｙ、１３、２１、１８号染色体     

应用荧光原位杂交技术快速诊断胎儿染色体数目异常

目的 探讨荧光原位杂交（ｆｌｕｏｒｅｃｅｎｔ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ,ＦＩＳＨ）技术在快速产前诊断胎儿染色体数目异常中的价值。方法 对２０例孕１６￣３６周,有产前诊断指征者,在Ｂ超引导下经腹抽取羊水后,应用Ｘ、Ｙ、１８号染色体着丝粒探针１３ｑ１４－ｑ２１和２１ｑ１１特异性探讨,对未培养的羊水间期细胞进行ＦＩＳＨ,然后用荧光显微镜进行观察,并用Ａｐｐｌｉｅｄ ｉｍａｇｉｎｇ染色体     

Role of FISH on uncultured amniocytes for the diagnosis of aneuploidies in the presence of fetal anomalies

OBJECTIVE: To assess the accuracy of fluorescent in situ hybridization (FISH) on amniocytes in fetuses affected by structural malformations suggestive of chromosomal anomalies. METHODS: FISH of uncultured amniotic fluid cells and conventional cytogenetic analysis were performed on 48 pregnancies with ultrasonographic (US) evidence of fetal anomalies. The AneuVysion assay (Vysis) with specific probes for chromosomes 13, 18, 21, X and Y, was used. Amniotic fluid samples were obtained between the 14th and 34th weeks of gestation. RESULTS: In cases with a single abnormal US finding (n = 15), 5 aneuploidies were detected (1 case of trisomy 13 and 4 of trisomy 21). In the group with two or more malformations (n = 33) there were 15 aneuploidies (9 cases of trisomy 18, 2 of trisomy 21, 2 monosomy X, 1 trisomy 13, and 1 triploidy). In this group, conventional cytogenetic analysis revealed two additional chromosomal anomalies not detectable by FISH (1 trisomy 16 mosaic, and a terminal deletion 4p). No sex aneuploidies were observed. CONCLUSIONS: The lack of false-positive diagnosis in the FISH analysis in our sample prompts us to consider interphase FISH as a useful tool in pregnancies at high risk for chromosomal aneuploidies. When FISH analysis is normal, the overall risk of chromosomal abnormalities is significantly reduced. However, the finding of two chromosomal anomalies undetectable by AneuVysion assay confirms the need for conventional chromosome analysis to complement FISH results. Moreover, the results collected here, in agreement with those already reported in the literature, indicate that FISH analysis on uncultured amniocytes can play an important role in counselling and decision-making, especially in cases at risk for aneuploidies, such as those with structural abnormalities at US.     

改良荧光原位杂交技术在产前诊断中的应用

目的:评价改良荧光原位杂交(fluorescent in situ hybridization,FISH)技术在产前诊断中的应用。方法:用改良FISH技术检测119例孕16~24周孕妇的羊水间期细胞及10例孕25~32周胎儿脐血间期细胞,5例孕9~12周绒毛间期细胞,每例均行常规染色体核型分析。结果:应用改良FISH法,所有样本均在6h内获得检测结果,除2例羊水培养失败外,其余样本均在3周内获得细胞遗传学诊断。两种方法均检出特氏综合征、18-三体综合征、21-三体综合征各1例,另5例常规染色体核型分析异常,因超出检测范围,FISH法未能检出,所有样本的两种方法检测结果均一致。结论:经改良后的FISH技术缩短了诊断时间,缓解了孕妇及家属的焦虑心情,且可用于多种不同样本的检测,因其高效、省时、取材多样等优点在产前诊断具有重要的临床价值。     

荧光原位杂交与核型分析技术产前诊断非整倍体的对比研究

目的:在羊水未培养细胞样本中建立并优化荧光原位杂交技术,应用国产探针检测,探讨其诊断未培养羊水细胞非整倍体的临床应用价值。方法:对100例孕12~27周未培养羊水细胞进行FISH快速产前诊断,对5条染色体(X、Y、13、21和18号)进行检测。以细胞培养常规核型分析作为FISH检测结果的对照。结果:被检100例羊水未培养细胞均获得诊断结果,检测出7例非整倍体,FISH结果与核型分析结果一致。结论:应用国产探针行FISH诊断未培养羊水细胞非整倍体,实验方法成熟稳定,结果准确可靠,符合产前诊断要求,有较大的临床应用价值。     

Prenatal Identification of a Tetraploid Fetus with fish

The first prenatal diagnosis of a tetraploid fetus via fluorescent in situ hybridization (FISH) technology is reported. FISH analysis of uncultured amniotic fluid cells utilizing probes for chromosomes 13, 21, 18, X, and Y revealed that greater than 65% of hybridized nuclei had four signals. Standard cytogenetic analysis revealed a 92, XXXX karyotype     

Rapid prenatal diagnosis of aneuploidies in uncultured amniocytes by fluorescence in situ hybridization. Evaluation of >3,000 cases.

OBJECTIVE: A new method in prenatal diagnostics allows to demonstrate certain numeric chromosomal aneuploidies in amniotic cells within 24 h in contrast to conventional methods which take 1-3 weeks. MATERIALS: The experience with this rapid fluorescence in situ hybridization (FISH) method is compared to standard karyotyping and its clinical relevance is described in a large clinical pilot study. FISH on uncultured amniocytes has been performed from 12 weeks of gestation to the third trimester using commercially available chromosome-specific DNA probes for chromosomes 13, 18, 21, X and Y. RESULTS: FISH was performed successfully in 3,150 prenatal cases. All trisomies 13, 18 and 21 and all cases with gonosomal aberrations were detected by FISH analysis. Neither false-positive nor false-negative results were obtained using FISH. For all analyzable disorders the FISH results were in complete agreement with standard cytogenetics. CONCLUSIONS: In our experience, FISH is a valuable and reliable method for rapid diagnosis of numeric chromosomal aneuploidies.     

Rapid prenatal diagnosis of trisomy 18 and triploidy in interphase nuclei of uncultured amniocytes by non-radioactive in situ hybridization.

Two biotinylated chromosome-specific DNA probes were used to quantify the number of chromosomes 18 and 1 in uncultured amniocytes. Thirty-three samples of uncultured amniocytes were hybridized with a chromosome 18-specific DNA probe. Uncultured cells from two of the 33 samples were also hybridized with a chromosome 1-specific probe. Thirty of the samples were disomic with respect to chromosome 18; two samples were trisomic with respect to chromosome 18, and one sample was trisomic with respect to chromosomes 1 and 18. The two cases of trisomy 18 and the single case of triploidy were identified on uncultured cells within 48-72 h after amniocentesis. They were found among five samples from pregnant women who had amniocentesis because of an ultrasonographically identified fetal malformation. A trisomic karyotype could be diagnosed with certainty in uncultured amniocytes because the majority of the responding nuclei exhibited three hybridization signals. In normal cells, the majority of nuclei exhibited two signals. In no cases was there discordance between the genotype as predicted by in situ hybridization and that determined by cytogenetic analysis.     

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.     

荧光原位杂交技术快速产前诊断胎儿血细胞非整倍体的价值

目的 探讨荧光原位杂交（FISH）技术在产前诊断脐血细胞非整倍体中的应用价值：方法 2004-06—2005-03，对广州市妇婴医院114例孕18～38W有产前诊断指征的孕妇进行脐血穿刺。采用X／Y染色体着丝粒探针和21q22．13-q22．2特异性探针对脐血细胞进行间期FISH检测，然后在荧光显微镜下观察，用Leica染色体核型自动分析仪QFISH软件进行图像的摄取和处理。同时所有脐血标本进行细胞培养，常规染色体G显带核型分析作为对照。结果 114例脐血标本都有FISH检测结果，107例具有正常核型染色体数目，异常7例，其中4例为唐氏综合征（3例为典型唐氏综合征，1例为嵌舍体），3例为性染色体数目异常。结论 FISH技术用于产前诊断脐血常见染色体数目异常，具有简便、快速、特异性强等优点，能为临床诊断提供依据。     

Rapid prenatal diagnosis of aneuploidy for chromosomes 21, 18, 13, and X by quantitative fluorescence polymerase chain reaction

OBJECTIVE: Quantitative fluorescence polymerase chain reaction (QF-PCR) is a rapid method for detection of chromosome copy number by amplification of repeat sequences at polymorphic loci. Our objective was to assess the performance of QF-PCR in detecting common aneuploidies in prenatal diagnosis. STUDY DESIGN: The study group consisted of pregnant women referred for amniocentesis or chorionic villus sampling (CVS) due to increased risk of fetal aneuploidy. Samples were collected from known affected and normal pregnancies. These were blindly screened for trisomy of chromosomes 21, 18, 13, and sex chromosome abnormalities, using QF-PCR. DNA from uncultured amniocytes was directly extracted using a modified alkaline lysis method. DNA from CVS was extracted by the phenol-chloroform procedure. Ten short tandem repeat (STR) markers were used for detection of fetal aneuploidy and gender. The STRs were selected for high heterozygosity rates and efficiency of the PCR amplification. The forward primer of each pair was labeled with a unique fluorescent dye. Amplified products were detected by an ABI Prism 310 Genetic Analyzer and results were analyzed using GeneScan Analysis Software. RESULTS: A total of 65 amniotic fluid and CVS samples were collected from affected and normal pregnancies. Two samples were contaminated with blood and were therefore excluded from the analysis. All 29 cases of aneuploidy were correctly diagnosed by QF-PCR, including 17 cases of trisomy 21, 7 cases of trisomy 18, and 5 cases with trisomy 13. The 34 normal samples were also correctly diagnosed as such. Thus, all results were in agreement with the standard cytogenetic results. There were no false-positive or false-negative results. CONCLUSION: We conclude that QF-PCR is a rapid, reliable, and reproducible method that may be used to provide rapid results in prenatal diagnosis of aneuploidy.     

Usefulness of fluorescence in situ hybridization for the diagnosis of Turner mosaic fetuses with small ring X chromosomes

OBJECTIVE: To emphasize the usefulness of fluorescence in situ hybridization (FISH) techniques on uncultured amniocytes for the diagnosis of abnormal mosaic karyotypes. METHODS: In the course of three prenatal diagnoses, specific fluorescent probes, coding, respectively, for chromosomes X, Y, 18, 13, and 21, were applied on amniocyte preparations directly after amniocentesis. At least 50 nuclei were counted in each case. Parallel to the FISH procedure, cell cultures were set up in order to obtain karyotypes. FISH and cytogenetic results were then compared. RESULTS: In each case, FISH showed an abnormal mosaic chromosomal constitution, 45,X/46,XX, which was related to the existence of tiny ring X chromosomes in karyotypes. CONCLUSION: Because very small ring X chromosomes can escape identification when standard cytogenetic techniques are used alone, we show that misdiagnosis can be avoided when FISH is performed beforehand.     

Elevated amniotic fluid leptin levels in pregnant women who are destined to develop preeclampsia

OBJECTIVE: To analyze whether leptin levels of the amniotic fluid elevate during early pregnancy in women destined to develop preeclampsia and to evaluate the relationship between amniotic fluid leptin levels and gestational age, maternal body mass index, and fetal sex. STUDY DESIGN: Leptin levels of the amniotic fluid were compared in two groups of women, preeclamptic (n = 20) and normotensive pregnant (n = 40), matched for fetal sex, maternal body mass index at sampling, gravidity and fetal gestational age at sampling. Furthermore, amniotic leptin levels in 400 normotensive pregnant women were analyzed for their correlation with gestational age, maternal body mass index, and fetal sex. RESULTS: Median leptin concentrations were significantly higher (p < 0.001) in the women with preeclampsia (7.3+/-0.7 ng/ml) than in the normotensive pregnant women (4.1 +/- 0.3 ng/ml), independent of fetal sex. The leptin levels in the amniotic fluid decreased with advanced gestational age (r = 0.24, p < 0.001). Amniotic fluid leptin levels in the pregnant women carrying a female fetus (5.6+/-0.3ng/ml) were significantly higher than those carrying a male fetus (4.7+/-0.2 ng/ml) (p = 0.004). CONCLUSION: Higher amniotic fluid leptin levels were observed in the preeclamptic pregnant women, and they decreased as gestational age advanced. Furthermore, the women with a female fetus were noted to have higher amniotic fluid leptin levels.     

The clinical application of interphase FISH in prenatal diagnosis

Fluorescence in situ hybridization (FISH) for five chromosomes (13, 18, 21, X and Y) detected 87 of 107 (81%) of the chromosome aberrations identified by conventional chromosome analysis applied to fetal interphase cells obtained by chorionic villus sampling or amniocentesis. The choice of FISH was solely determined by prospective parents after formal genetic counselling concerning the advantages and disadvantages of FISH analysis. Excluding known familial chromosome aberrations, if FISH analysis revealed normal signals, there was an overall residual risk of 1 in 149 for an undetectable chromosome aberration. This risk varied according to the indication for prenatal diagnosis: 1 in 177 for women of advanced maternal age; 1 in 60 for women at increased risk for Down syndrome based on maternal serum screening; and, 1 in 43 for women whose ultrasound examination revealed fetal anomalies. There were 20 cases of discordance between the FISH results and standard karyotype analysis: four were the outcome of a failure to apply the appropriate FISH probe; 16 were not detectable by the available FISH probes. Of these 16, nine were chromosome abnormalities with clinical significance and seven were familial. If FISH is to become a standard part of prenatal genetic diagnosis, genetic counselling that is sensitive to patient health needs must be based on accurate information about the biological and obstetrical implications of the results of FISH analysis.     

Familial X centromere variant resulting in false-positive prenatal diagnosis of monosomy X by interphase FISH.

Interphase fluorescent in situ hybridization (FISH) analysis performed on uncultured amniotic fluid cells from a female fetus revealed a single signal using an X chromosome alpha-satellite probe, and the absence of any signal using a Y chromosome alpha-satellite probe. This result was initially interpreted as monosomy for the X chromosome in the fetus. Subsequent chromosome analysis from the cultured amniotic fluid cells showed two apparently normal X chromosomes. FISH using the X alpha-satellite probe on metaphase spreads revealed hybridization to both X chromosomes, although one signal was markedly reduced compared to the other. The same hybridization pattern was observed in the mother of the fetus. This is the first report of a rare familial X centromere variant resulting in a false-positive diagnosis of monosomy X by interphase FISH analysis for prenatal diagnosis.     

Simultaneous Detection of Chromosomes X, Y, 13, 18, and 21 by Fluorescence In Situ Hybridization in Blastomeres Obtained from Preimplantation Embryos

Purpose. Simultaneous fluorescence in situ hybridization (FISH) was used in a preimplantation genetic diagnosis program to determine which embryos were normal for chromosomes X, Y, 13, 18, and 21. Methods: Single blastomeres were disrupted and attached to glass slides using acetic acid and ethanol. Using a ratio mixture of chromosome enumeration DNA probes in combination with locus-specific identifier DNA probes, FISH was performed for the identification of chromosomes X, Y, 13, 18, and 21. Results: Fourteen couples enrolled in IVF produced 134 embryos for biopsy. Blastomeres subjected to five-color FISH revealed that 22% of embryos were normal for chromosomes X, Y, 13, 18, and 21. In addition, 52% were abnormal and no results could be detected for 25%. Twelve couples underwent embryo transfer, two couples did not receive embryos due to lack of any normal embryos, and three couples became pregnant. Conclusions: The simultaneous detection of five-color FISH is a feasible method to detect aneuploidy in preimplantation embryos from women of advanced maternal age.     

无创DNA检测在诊断高龄孕妇胎儿非整倍体中的应用

目的:探讨高龄妊娠胎儿染色体异常的风险以及无创DNA产前检测(NIPT)在诊断高龄孕妇胎儿非整倍体染色体病中的应用价值。方法:选择≥35岁的高龄孕妇2714例,按年龄分为35~39岁,≥40岁两组,采用NIPT高通量测序检测孕妇血浆游离DNA,并对检测结果提示21-三体、18-三体、13-三体及性染色体高风险者行羊膜腔穿刺术及胎儿染色体核型分析,对检测结果阴性者通过电话随访进行验证。计算NIPT检测的敏感度、特异度、阳性预测值、阴性预测值及Youden指数。结果:2714例高龄孕妇NIPT检测结果提示胎儿非整倍体染色体异常高风险47例,6例高风险孕妇拒绝侵入性产前诊断,余41例高风险孕妇行羊膜腔穿刺术及羊水细胞染色体核型分析,结果显示21-三体19例,18-三体1例,13-三体2例,性染色体异常7例。与现有的金标准羊膜腔穿刺术核型分析相比较,NIPT对高龄孕妇除外性染色体异常的胎儿非整倍体染色体异常检出敏感度为100.00%,特异度为99.93%,阳性预测值为90.91%,阴性预测值为100.00%,Youden指数为0.99。进一步通过年龄分组发现,40岁及以上年龄组异常率显著高于35~39岁年龄组(P=0.011)。结论:高龄孕妇可通过NIPT快速、安全地筛查出胎儿非整倍体染色体异常,减少侵入性产前检测比例,降低胎儿出生缺陷率。     

Effectiveness of prenatal chromosomal analysis using multicolor fluorescent in situ hybridisation

Objective To evaluate the clinical effectiveness of multicolour fluorescent in situ hybridisation (FISH) analysis in routine prenatal diagnosis.
Design Prospective study.
Sample 3203 amniotic fluid samples.
Methods Unique DNA (chromosomes 13 and 21) and α satellite centromeric-specific (chromosomes X, Y and 18) probes were used in two mixes to permit the simultaneous analysis of several chromosomes. The performance of multicolour FISH and conventional cytogenetic analysis was compared.
Results Conventional cytogenetic analysis identified 111 chromosomal abnormalities, of which 94 were potentially detectable by the FISH technique and 97 would be typically associated with neonatal phenotypic abnormalities. Multicolour FISH analysis detected 84% (93/111) of all chromosome abnormalities and 99% (93/94) of abnormalities where there was a specific probe. The sensitivity of multicolour FISH analysis was 95% (92/97) for chromosomal abnormalities likely to result in an abnormal postnatal outcome. Multiple ultrasound abnormalities were detected in all five cases of clinically relevant chromosomal abnormalities missed by multicolour FISH. FISH results were available within 48 hours and the sample failure rate was 0.1% (3/3202).
Conclusion Multicolour FISH analysis is a sensitive and reliable technique for the rapid prenatal diagnosis of chromosomal abnormalities. Examining only five chromosomes allowed 95% of clinically relevant chromosomal abnormalities to be diagnosed correctly. As routine antenatal screening is targeted at the major autosomal trisomies and sex chromosome aneuploidies, multicolour FISH analysis may potentially replace conventional cytogenetic analysis in routine prenatal diagnosis.     

Risk of false-positive prenatal diagnosis using interphase FISH testing: hybridization of alpha-satellite X probe to chromosome 19.

FISH analysis of uncultured interphase amniotic fluid cells from a male fetus revealed two signals using an alpha-satellite X-chromosome DNA probe. One of the signals was much smaller than the other. It was subsequently shown that the normal sized signal was located on the X chromosome and the smaller signal was located at the centromere of chromosome 19. This hybridization pattern was confirmed in the newborn infant and in his phenotypically normal father. The use of alpha-satellite DNA probes on interphase cells could result in false-positive errors due to rare variants such as the X-chromosome alpha-satellite found on chromosome 19 in our patient.     

Follow-up investigations in uncultured amniotic fluid cells after uncertain cytogenetic results

Fluorescence in situ hybridization (FISH) on uncultured amniotic fluid (AF) cells is a widespread technique for the rapid prenatal detection of specific chromosome aberrations. During a 6-year period (1993-1998) we used FISH for quick follow-up investigations in uncultured AF cells after finding an uncertain chromosome aberration in a first chorionic villus (CV) or AF sample in 79 cases. These FISH results were compared with conventional cytogenetic results of the AF cell cultures in all cases. We found discrepant FISH and cytogenetic results in four instances. In general, FISH on uncultured AF cells proved to be a reliable technique for the rapid differentiation between confined placental mosaicism and true fetal mosaicism, and between pseudomosaicism and true mosaicism, respectively. Uncultured cells may sometimes even better reflect chromosomal mosaicism than cultured cells, since they are not subject to culture induced selection mechanisms. However, we found evidence that exceptional cases of tissue confined mosaicism may go undetected in uncultured cells.