唐氏综合征产前羊水诊断补救措施的探讨

目的对错过唐氏筛查和羊水胎儿细胞染色体核型分析的孕妇进行唐氏综合征产前诊断补救措施的探讨与研究。方法抽取首都医科大学附属北京妇产医院就诊的孕妇羊水3 ml共103例,均已诊断胎儿异常（染色体异常或大体畸形等）,提取羊水中胎儿细胞DNA,应用QF-PCR方法进行唐氏综合征的分子诊断。结果诊断周期48～72 h,诊断准确率达95%,孕周24～35周,年龄21～44岁,所有患者均进行了胎儿染色体的核型分析和QF-PCR法分析,其中9例为唐氏综合征胎儿。结论QF-PCR方法产前诊断妊娠24周以上的孕妇,与脐静脉穿刺血细胞培养染色体核型分析诊断相比,更安全,简便,快速,依从性好（妊娠24周以上已经不能进行羊水的胎儿细胞培养和核型分析）。     

实时荧光定量聚合酶链反应技术在产前诊断唐氏综合征中的应用

目的探讨实时荧光定量PCR技术用于产前诊断唐氏综合征的可行性。方法采用实时荧光定量PCR法,分别检测85例唐氏综合征高风险的中期妊娠妇女的羊水和7例智残儿外周血标本中,21号染色体上特异区域基因片段(DSCR3)和管家基因片段(GAPDH),并计算两者的比值。同时采用细胞遗传学方法分析其染色体核型。结果80例羊水细胞DNA检测DSCR3／GAPDH比值在0．46—1．30之间,染色体核型全部正常;而另5例羊水细胞和7例智残儿外周血DSCR,／GAPDH比值明显升高,达1．64～1．98,染色体核型均为21三体。5例中有3例核型为47,XY, 21;1例核型为47,XX, 21;另1例为易位型[46,XY,-15, t(15;21)]。7例智残儿中4例为47,XY 21;3例为47,XX 21。结论实时荧光定量PCR技术可作为产前快速准确诊断唐氏综合征的有效方法。     

15230例孕中期唐氏筛查产前诊断的临床应用

目的:探讨孕中期唐氏筛查对检出胎儿染色体异常的预测价值。方法:2008年1月至2009年10月,采用时间荧光免疫分辨法对我院15230例孕中期(15～20+6周)妇女进行血清标志物甲胎蛋白(AFP)、游离雌三醇(uE3)、绒毛膜促性腺激素(β-HCG)3项指标进行检测,对于筛查结果为高风险的孕妇于孕20～24周行羊膜腔穿刺进行胎儿羊水细胞染色体核型分析,并对唐氏筛查情况进行效果评价。结果:984例孕妇唐氏筛查为高风险,高风险率为6.46%,其中唐氏综合征阳性孕妇736例,18-三体阳性78例,神经管缺陷阳性169例。有773例高风险孕妇接受羊水穿刺,发现胎儿染色体异常29例,异常检出率为3.75%,其中唐氏综合征11例,18-三体1例,69,XXX1例。唐氏筛查的敏感性和特异性分别为92.86%和95.25%。结论:孕中期唐氏筛查是预测异常胎儿和不良妊娠结局的有效手段之一,羊水细胞核型分析在产前诊断中具有重要的实用价值。     

STR-PCR在产前诊断胎儿唐氏综合征中的应用

目的:研究通过多重荧光定量PCR诊断胎儿染色体非整倍体用于临床快速产前诊断的可行性。方法:从孕中期羊水中提取胎儿DNA,通过多重荧光定量PCR使用STR对13、18、21号染色体进行非整倍体筛查,筛查结果异常者再进行快速诊断。用PCR诊断的羊水标本同时使用"金标准"染色体核型分析法做对比。结果:34例羊水标本中2例标本由于母血污染严重未行PCR检测,1例标本经PCR及核型分析均失败,29例标本经PCR和核型分析诊断为正常染色体,2例标本经PCR和核型分析诊断为21-三体。结论:通过STR-PCR法使用多重荧光酶联聚合反应探针产前诊断胎儿唐氏综合征是临床快速产前诊断的有效方法之一。     

应用荧光原位杂交产前诊断染色体异常的临床价值

目的:探讨应用荧光原位杂交(HSH)产前诊断染色体非整倍体的临床价值.方法:收集120例产前诊断孕妇的新鲜羊水进行FISH检测和染色体核型分析,并将结果与临床追踪确诊结果(随访的新生儿或引产的死胎脐血或外周血的染色体核型)作比较,同时根据FISH的检测效能和分析产前诊断方案,评价HSH的临床应用价值.结果:①HSH检测全部成功,其结果与临床追踪确诊的核型分析一致,并且染色体非整倍体检出率100%;1例孕晚期羊水细胞培养失败,2例羊水培养为四倍体镶嵌体胎儿经临床追踪确诊后为正常染色体.②产前诊断指征中,高龄、多项指征及其他因素的孕妇临床上对FISH及核型分析这两种方法的选择比较,差异无统计学意义(P>0.05);而血清唐氏筛查异常和超声筛查异常的孕妇分别倾向选择FISH(P=0.029)及核型分析(P=0.000).结论:HSH技术能快速准确检测染色体非整倍体的异常.母血清唐氏筛查异常孕妇产前诊断倾向选择FISH检测.FISH可作为孕晚期高危孕妇首选的产前诊断方法.     

3657例孕中期唐氏筛查及产前诊断的临床价值分析

目的探讨孕中期唐氏筛查对检出胎儿染色体异常和妊娠不良结局的临床价值。方法应用时间分辨荧光免疫法对3657例孕中期(14～20+6周)妇女进行血清标记物AFP和free-β-hCG2项指标双标记检测。筛查结果应用LifeCycle和Elipse软件计算唐氏综合征风险。唐氏筛查风险切割值为1∶270,当切割值≥1∶270时为唐氏高危孕妇。对于高危孕妇,于孕18～22周左右进行羊膜腔穿刺,抽取羊水进行胎儿染色体核型分析。并继续追踪胎儿和孕妇情况。结果在有回访资料的3258例孕妇中,去筛查到高危212例,唐氏筛查阳性率为5.8%(212/3657)。其中68例接受羊水或脐血穿刺产前诊断,占筛查高危孕妇的32.1%(68/212);发现胎儿染色体异常8例,异常检出率11.8%(8/68),其中3例唐氏综合征、2例18-三体综合征、1例Turner’s综合征、1例9号染色体臂间倒位、1例平衡易位。唐氏筛查高风险和低风险组不良妊娠结局分别为6.6%和3.4%,呈显著性差异(p0.05)。结论孕中期产前筛查是预测异常胎儿和不良妊娠结局的有效指标。结合羊水培养或脐血培养等产前诊断技术和方法,对预防先天缺陷儿出生有重要临床应用价值。     

利用PRINS技术进行无创性产前诊断胎儿非整倍体

目的利用引物原位合成术(primedin situ labelling,PRINS)进行产前诊断胎儿非整倍体,探索无创性产前诊断的可靠便捷的新方法。方法采用流式细胞术从120例孕妇外周血中分离出胎儿有核红细胞,应用PRINS技术分别检测胎儿有核红细胞内的X、Y及21号染色体。结果120例标本中每例都可以检测出X染色体,敏感性和特异性均为100%;检测出Y染色体69例,敏感性92%(69/75),特异性为100%(69/69),检测出Klinefelter(XXY)综合征1例,唐氏综合征2例。结论应用PRINS技术对孕妇外周血中的胎儿细胞进行无创性产前诊断胎儿非整倍体是一种快速、敏感性高、特异性强的新方法,具有应用于临床的前景与价值。     

从孕妇外周血分离滋养细胞和胎儿有核红细胞作非侵入性产前诊断

非侵入性快速胎儿核型分析需从孕妇外周血中分离胎儿细胞和用荧光原位杂交检测染色体。用特异性单克隆抗体与磁珠交联,结合梯度离心和微磁激活细胞分离法从孕妇外周血中富集到滋养细胞和/或胎儿有核红细胞以诊断胎儿异常。 研究对象为孕10～14周行绒毛活检的孕妇41例,每例取肝素化静脉血15～20ml,离心后弃血清,加入无血清培养液,再加入与磁珠交联的可识别滋     

荧光原位杂交技术在未培养羊水细胞产前诊断中的应用研究

目的:探讨运用荧光原位杂交(FISH)技术对未培养羊水细胞的染色体非整倍体进行快速诊断的临床应用价值.方法:对符合产前诊断要求的孕妇于孕18～24周抽取羊水,用13、21、18及X、Y号染色体探针对其未培养羊水细胞进行诊断,并与核型分析结果比较.结果:125例标本的核型分析中,成功培养123例,其中117例为正常核型,6例为异常核型,异常核型为21-三体2例,9号染色体臂间倒位3例,6号和7号染色体平衡易位1例;与核型分析相比,FISH只检测出2例非整倍体核型,其余染色体结构异常未能检出,FISH检测时间为24～48小时,较传统核型分析时间2～3周大大缩短,且同核型结果一致,特异性100%.结论:荧光原位杂交技术具有简便、准确等优点,可快速地辅助核型分析,缓解患者及家属情绪,有临床推广价值.     

胎儿染色体核型异常3例报道

为了降低围生儿死亡率 ,减少出生缺陷儿的发生 ,我们自 2 0 0 0年 11月 1日至 2 0 0 1年 7月 3 1日对沈阳市九区的6664例妊娠 14～ 2 0周的孕妇进行了产前胎儿核型异常的筛查。经外周血监测ＡＦＰ、ＦＲＥＥ β ＨＣＧ疑有胎儿核型异常的高危孕妇共 417例 ,在医生的建议下 ,有 10 0例夫妇同意羊膜腔穿刺抽取羊水进一步确诊 ,经羊水细胞培养及荧光原位杂交 (ＦＩＳＨ)进行胎儿染色体核型分析 ,诊断胎儿异常有 3例。分别为 18三体 2例 ,克氏症 1例 ,均已行治疗性流产。现将 3例胎儿染色体核型异常报道如下。例 1.孕妇 3 3岁 ,孕 3产 0 ,非近亲…     

Examination of fetal cells and cell-free fetal DNA in maternal blood for fetal gender determination

BACKGROUND: To assess applicability of noninvasive methods for prenatal sex determination, both intact fetal cells and cell-free DNA from maternal blood were studied. METHODS: Maternal peripheral blood samples were obtained from 41 women carrying chromosomally normal fetuses and from 3 women with aneuploid fetuses (47,XX,+18; 47,XY,+18 and 47,XY,+21) at 9-22 weeks of gestation. DNA was extracted from the plasma fraction and analyzed by the nested polymerase chain reaction (PCR) using Y chromosome specific primers. After fetal cells were enriched by MACS, fluorescence in situ hybridization (FISH) with chromosome X and Y specific probes was performed to detect XY cells. RESULTS: Although Y-chromosome-specific DNA was detected by PCR analysis in all maternal plasma samples with male fetuses, 26% women bearing female fetuses also gave positive results. By FISH analysis, XY cells were detected in not only 58% of women bearing male fetuses, but also 13% of their counterpoints with female fetuses. CONCLUSIONS: Our findings suggested that consistent results for fetal gender using PCR or FISH cannot be obtained with intact fetal cells and cell-free DNA present in maternal blood and plasma at 9-22 weeks of gestation, despite their apparent abundant presence.     

Quantification of fetal nucleated cells in maternal blood of pregnant women with a male trisomy 21 fetus using molecular cytogenetic techniques

BACKGROUND: Prenatal diagnosis of trisomy 21 is based on fetal karyotyping generally obtained using invasive methods. During pregnancy, the circulating fetal cells in maternal blood constitute a potential source for development of a noninvasive prenatal diagnosis. The objective of this study was the identification and quantification of all fetal nucleated cells per unit volume of peripheral blood of pregnant women carrying male fetuses with trisomy 21 using molecular cytogenetic techniques. METHODS: Peripheral blood samples were obtained from 16 women carrying male fetuses with trisomy 21. We used a simple and rapid method of harvesting blood without recourse to any enrichment procedures or cell-separation techniques. To evaluate the potential of this method, 16 specimens were analyzed by molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) using specific probes to chromosomes X, Y and 21. RESULTS: The number of fetal cells varied between 6 and 32 per mL of maternal blood. This number is 3-5 times higher than that from normal pregnancies. CONCLUSIONS: Our current results are in agreement with the results previously reported by other groups showing that the number of fetal cells in maternal blood in trisomic 21 pregnancies is higher than in normal pregnancies. This high number of fetal cells is regarded as an advantage for the development of a noninvasive prenatal diagnostic test.     

富集孕妇外周血中胎儿细胞行胎儿性别诊断

目的探讨对孕妇外周血中胎儿细胞进行非侵入性产前诊断的可行性。方法对６４例孕８～４０周孕妇外周血进行密度梯度离心法,富集胎儿细胞,并经显微镜下观察计数各种细胞所占百分比。同时,对所富集细胞经提取ＤＮＡ进行人Ｙ染色体特异ＤＮＡ扩增,判定胎儿性别并与新生儿性别进行对照。结果６４例孕妇外周血中,２５例观察到有核红细胞,占３９．０６％。６４例孕妇分娩３３例男性婴儿,其中２８例扩增出Ｙ特异带;另外３１例分娩女性婴儿,除１例出现Ｙ特异带,其余未见特异带。诊断的灵敏度为８４．８５％,特异度为９６．７７％,总符合率为９０．６３％。结论密度梯度离心法可从孕妇血中富集胎儿细胞,且所获得细胞已基本满足体外扩增Ｙ染色体基因所需要的模板量。     

Fetal nucleated red blood cells in peripheral blood of pregnant women: detection and determination of location on a slide using laser-scanning cytometry

OBJECTIVE: The purpose of the study was to assess the feasibility of analysis of fetal nucleated red blood cells (NRBC) present in the maternal circulation by laser-scanning cytometry. METHODS: CD71-positive cells were obtained by magnetic cell sorting of peripheral blood of pregnant women after density centrifugation. Immunofluorescence for the Hbgamma-chain was combined with fluorescent staining of DNA (TO-PRO-3) and fluorescence in situ hybridization (FISH) with a Y-chromosome specific probe. The cells were scanned on a slide using a laser-scanning cytometer (LSC). Events double positive for Hbgamma and TO-PRO-3 were relocated and their morphology and FISH reactivity were visually assessed. Determination of male fetal sex with LSC was compared with findings from amniocentesis. RESULTS: In 8/15 pregnancies with male fetuses and in 0/9 with females (apart from one case with a male/female twin pregnancy), we detected Y-chromosome-positive NRBC. In pregnancies with female fetuses, Y-chromosome-positive cells other than NRBC were found in all women who had previously given birth to male babies, whereas women with no abortion and no male babies in their history did not present with Y-chromosome-positive non-NRBC. CONCLUSION: On the basis of automatic relocation of once-defined cells of fetal origin from the current pregnancy, laser-scanning cytometry is likely to facilitate repeated (poly-)FISH analysis and single-cell PCR for noninvasive prenatal diagnosis.     

Quantitative FISH analysis and in vitro suspension cultures of erythroid cells from maternal peripheral blood for the isolation of fetal cells

Several techniques for the enrichment of nucleated fetal red blood cells present in maternal blood have been reported. Here we describe the use of a quantitative fluorescence in situ hybridization (FISH) method and in vitro suspension cultures of erythroid cells from newborn cord blood and maternal peripheral blood. Together with a rapid high performance liquid chromatography (HPLC) method, that allows us to determine as few as 100 cells containing haemoglobin F (HbF), we have scrutinized the reported enrichment methods for fetal nucleated cells in peripheral maternal blood. One hundred FISH analyses on maternal peripheral blood were performed. The method comprises a cell lysis method for depletion of red cells with minimal losses of nucleated cells, uniform numbers of cells (750 000 cells each) on microscopic slides, and inclusion of internal controls to monitor the efficacy of hybridization. Twenty-six cultures of pure erythroid progenitor cells from maternal peripheral blood were analysed for the expansion of fetal cells. To generate these in vitro cultures, nucleated cells from 10-20 ml of peripheral blood from 26 pregnant women were grown in media containing growth factors and hormones to yield over 10(7) of immature erythroid cells within two weeks. Of those, 13 cultures were from pregnancies with confirmed male fetuses. A total of approximately 8x10(8) maternal cells were added into tissue culture medium for these 13 cultures, resulting in about 2x10(8) nearly pure erythroid cells after two weeks. Whereas fetal cells, alone or added into cultures of peripheral blood, grow rapidly and can be detected quantitatively, we could not find any fetal cells in cultures from maternal blood. Likewise, in 7.5x10(7) peripheral blood cells probed by FISH analysis (half of which were from pregnancies with male fetuses) no single Y chromosome was detected. In summary, suspension cultures of erythroid cells can be established routinely and easily. With the quantitative FISH technique used, 750 000 cells per slide can be screened reliably for cells with Y chromosomes. However, the stringent quality-criteria and most elaborate methods indicate that fetal cells in maternal peripheral blood can not be found using the current technology.     

Enrichment, identification and analysis of fetal cells from maternal blood: evaluation of a prenatal diagnosis system.

In this study we evaluated the performance of a system for the enrichment, identification and analysis of fetal cells in maternal peripheral blood. Blood samples were collected from women after chorionic villus sampling and enriched for the presence of nucleated erythrocytes using a three-step procedure, namely: (a) centrifugation to separate nucleated red blood cells (NRBCs) from the majority of red blood cells (RBCs) and white blood cells (WBCs); (b) selective lysis of the remaining maternal RBCs; (c) separating the NRBCs from the remaining WBCs in a three-layer density gradient. Fetal cells were identified by using a monoclonal antibody against the gamma-chain of fetal haemoglobin (anti-HbF) and a nuclear stain (DAPI). Additionally, to further increase the specificity of the identification, and to eliminate some of the undesired staining by maternal leukocytes, a fluorescent antibody (CD45) was added. The sex chromosome complement of the cells was determined by fluorescence in situ hybridization (FISH) with X and Y-specific probes and the results were compared with the karyotypes obtained after analysis of chorionic villi. Using the described method, in all cases where the woman was carrying a male fetus (n=18) at least one XY cell was found, while no male cells were found in women carrying a female fetus. However, in the majority of cases with a male fetus (n=11) female HbF positive cells were found indicating the presence of maternal nucleated erythrocytes. The study demonstrates that the combination of anti-HbF and CD45 is a useful, but not fully specific, marker for fetal NRBCs and that additional markers are needed.     

No correlation between the number of fetal nucleated cells and the amount of cell-free fetal DNA in maternal circulation either before or after delivery

OBJECTIVES: We have determined the number of fetal nucleated cells and the concentration of cell-free fetal DNA in parallel in the same maternal blood samples either before or after delivery, and studied the relationship between these two. METHODS: Venous blood samples were taken at four points around delivery from ten women who had singleton male fetus with informed consent. The number of fetal nucleated cells having a Y chromosome specific signal treated by two-color fluorescence in situ hybridization technique was counted using maternal whole blood. The concentration of sex-determining region Y gene sequence was determined using real-time quantitative PCR. RESULTS: The number of fetal nucleated cells decreased after delivery, and some fetal cells were present 1 month after delivery. While cell-free fetal DNA decreased rapidly after delivery and became undetectable 1 day after delivery in eight out of ten cases. The number of fetal nucleated cells did not correlate with the concentration of cell-free fetal DNA in maternal circulation. CONCLUSION: The present study demonstrates that cell-free fetal DNA disappears very rapidly after delivery and fetal nucleated cells remain longer in maternal circulation, and that there is no correlation between these two either before or after delivery.     

Non-invasive prenatal diagnosis by isolation of both trophoblasts and fetal nucleated red blood cells from the peripheral blood of pregnant women

Objective To isolate fetal trophoblasts and nucleated red blood cells from the peripheral blood of pregnant women.
Design Trophoblasts were isolated from whole blood of women in the first trimester of pregnancy by a specific monoclonal antibody, 340. Nucleated red blood cells were isolated by separating whole blood on a triple gradient, staining with ferromagnetic particles coated with an antitransferrin monoclonal antibody and separated on a mini magnetic activated cell sorting (MACS) column. Sorted cells were sexed using a nested polymerase chain reaction for a specific sequence on the Y chromosome and the sex was confirmed by karyotyping of chorionic villus samples.
Participants Patients between 10 and 14 weeks of pregnancy who were undergoing elective chorionic villus sampling for the detection of fetal aneuploidies.
Main outcome measure Fetal sex determined by polymerase chain reaction on fetal cells sorted from maternal blood.
Results When both trophoblasts and nucleated red blood cells were sorted, fetal sex was correctly predicted in 12/13 cases (92%), which included correct diagnosis of five of six male pregnancies. More importantly the two techniques were complementary, with only one male pregnancy being diagnosed on both trophoblasts and nucleated red blood cells, two being detected only with trophoblasts and two on nucleated red blood cells alone. No false positives (male signal from a female pregnancy) were diagnosed with either trophoblasts or nucleated red blood cells even with the highly sensitive nested polymerase chain reaction technique, which is very prone to contamination. This study also shows that it is possible to isolate both trophoblasts and nucleated red blood cells from the same sample of maternal blood.
Conclusion Fetal cells can be isolated from maternal blood at around 10 weeks of pregnancy.     

Detection of gamma-globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization

OBJECTIVES: Fetal nucleated red blood cells (NRBC) that enter the peripheral blood of the mother are suitable for non-invasive prenatal diagnosis. The application of peptide nucleic acid (PNA) probes for tyramide amplified flow fluorescence in situ hybridization (FISH) detection of gamma-globin mRNA in fixed fetal NRBC is investigated. METHODS: Hemin-induced K562 cells or nucleated blood cells (NBC) from male cord blood were mixed with NBC from non-pregnant women and analysed using both slide and flow FISH protocols. Post-chorionic villus sampling (CVS) blood samples from pregnant females carrying male fetuses were flow-sorted (2 x 10(6) NBC/sample). Y chromosome-specific PNA FISH was used to confirm that the identified gamma-globin mRNA stained cells were of fetal origin. RESULTS: Flow FISH isolated gamma-globin mRNA positive NBCs showing characteristic cytoplasmic staining were all Y positive. The amplification system generated a population of false positive cells that were, however, easy to distinguish from the NRBCs in the microscope. CONCLUSION: The gamma-globin mRNA specific PNA probes can be used for detection and isolation of fetal NRBCs from maternal blood. The method has additional potential for the study of gamma-globin mRNA levels or the frequency of adult NRBC (F cells) in patients with hemoglobinopathies.     

Significantly higher number of fetal cells in the maternal circulation of women with pre-eclampsia.

Although the pathophysiology of pre-eclampsia is unknown, several studies have indicated that abnormal placentation early in pregnancy might play a key role. It has recently been suggested that this abnormal placentation may result in transfusion of fetal cells (feto-maternal transfusion) in women with pre-eclampsia. In the present study, fetal nucleated red blood cells were isolated from 20 women with pre-eclampsia and 20 controls using a very efficient magnetic activated cell sorting (MACS) protocol. The number of male cells was determined using two-color fluorescence in situ hybridization (FISH) for X and Y chromosomes. Significantly more XY cells could be detected in women with pre-eclampsia (0.61+/-1.2 XY cells/ml blood) compared to women with uncomplicated pregnancies (0.02+/-0.04 XY cells/ml blood) (Mann-Whitney U-test, p<0.001). These results suggest that fetal cell trafficking is enhanced in women with pre-eclampsia, and this finding may contribute to the understanding of the pathophysiology of the disease.