荧光定量PCR检测Rh阴性孕妇外周血浆游离DNA中胎儿RhD血型

目的 探讨荧光定量PCR(fluorescence quantitative polymerase chain reaction,FQ-PCR)技术对Rh阴性孕妇血浆中游离胎儿DNA进行非创性产前诊断胎儿RhD血型的可行性.方法 选取78份妊娠11～40周、B超确诊为单胎的Rh阴性孕妇血浆.采用9个短串联重复序列(short tandem repeat,STR)多态性位点及Y染色体性别决定区基因(sex-determining region Y chromosome,SRY)确定胎儿DNA的存在;运用FQ-PCR技术对血浆中游离胎儿DNA进行RHD基因外显子5、7、10和内含子4定量分析,以确定胎儿RhD血型的基因型;其基因型结果与产后新生儿脐血血清学检测结果进行对比分析,回顾性评价胎儿基因定型结果的准确性.结果 78份标本中,41份检测到SRY基因,平均浓度为(214.7±120.9)拷贝/ml,产后证实皆为男性.70份FQ-PCR基因定型结果与血清学结果相符,另有5份确定为假阳性,3份基因定型结果不可确定,检测结果总符合率为90%(70/78).5份假阳性标本通过检测RHD1227A等位基因鉴定了4份RhD放散型,FQ-PCR最终结果准确率达到95%(74/78).结论应用FQ-PCR方法进行非创性胎儿RhD血型检测可用于新生儿溶血病的预防和诊断.     

PCR-SSP技术对羊水细胞ABO血型的基因鉴定

目的通过PCR-SSP基因技术检测胎儿羊水细胞ABO血型基因型,产前诊断胎儿ABO血型。方法选取了6名孕16 W以上的孕妇,抽取羊水细胞并进行分离,提取羊水细胞DNA,运用PCR-SSP技术分析其ABO血型基因型,并通过出生后的脐带血的血型鉴定进行确认。结果 6例羊水标本均通过PCR-SSP方法检测出了ABO血型的基因型;该6名胎儿的脐带血的ABO血型与羊水细胞的血型一致。结论 PCR-SSP技术可以准确地检测胎儿羊水细胞的ABO血型。     

孕妇血浆游离DNA检测在母婴ABO血型不合诊断中的临床研究

目的 探索一种利用孕妇血浆中游离DNA检测胎儿ABO血型用于母婴ABO血型不合的无创性产前诊断方法.方法 用硅胶膜柱提取的方法从50例13～37孕周可疑母婴ABO血型不合的孕妇血浆中提取胎儿DNA,采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法检测胎儿ABO血型.结果 50例样本中,检出32例,检出率64%(32/50),出生后血清学证实28例诊断正确,诊断正确率87.5%(28/32).结论 利用孕妇血浆中游离DNA检测胎儿ABO血型可行,对诊断和预防因母婴ABO血型不合所致溶血病的发生具有重要意义.     

荧光定量PCR在乙型肝炎病毒宫内感染监测中的应用

目的 探讨荧光定量聚合酶链反应(FQ-PCR)在乙型肝炎病毒(HBV)宫内感染监测中的作用及临床意义.方法 应用FQ-PCR检测102例乙型肝炎(下称乙肝)感染孕妇血清及其新生儿脐带血的HBV DNA,应用酶联免疫吸附试验(ELISA)检测乙肝感染孕妇血清及其新生儿脐带血的乙肝血清学标志物.结果 新生儿脐带血的HBVDNA阳性率为16.7%(17/102),高于脐带血乙肝病毒表面抗原(HBsAg)的阳性率(10.8%,11/102)P＜0.001;102例乙肝孕妇中有29例血清乙型肝炎病毒e抗原(HBeAg)阳性,其新生儿脐带血HBV DNA阳性率为48.3%(14/29),高于孕妇HBeAg阴性组的阳性率(4.1%,3/73)P＜0.001,新生儿脐带血的HBV DNA阳性率随孕妇HBVDNA含量增加而增加(P＜0.001).结论 FQ-PCR检测新生儿脐带血HBV DNA是诊断乙肝宫内感染的良好而敏感的筛选指标;孕妇血清HBV DNA高含量是乙肝宫内感染的高危因素.     

全自动血型仪在AB0及Rh(D)血型检测中的应用

目的:对全自动血型仪在血站系统中检测ABO血型及Rh(D)血型的结果进行分析.方法:统计分析2005-11/2006-05应用全自动血型仪检测献血者ABO血型及Rh(D)血型后所得结果的正确率、正反不符率、错误率.结果:13 854例献血者ABO血型结果的正确率为99.1%,正反不符率0.9%,错误率0%.Rh(D)血型结果的正确率为100%.结论:全自动血型仪可用于献血者ABO血型及Rh(D)血型的检测.     

渝东北地区胎儿染色体非整倍体疾病的NITP分析

目的应用无创产前基因检测(noninvasive prenatal genetic testing,NIPT)技术研究渝东北地区胎儿染色体非整倍体疾病的发病率和年龄分布。方法利用美国life公司二代测序平台及生物信息分析技术,分析2016年7月～2018年5月在重庆三峡中心医院基因检测中心行NIPT检测的2 784例12～24周孕妇的外周血,从中获取胎儿染色体相关信息。对NIPT结果为高风险的孕妇实施羊膜腔穿刺及胎儿染色体核型分析,对NIPT结果为低风险的孕妇进行电话随访至出生后。结果渝东北地区孕妇染色体非整倍体疾病高风险阳性检出率为0.72%(20/2 784)。其中≥40岁阳性率为2.97%,30～39岁阳性率为0.75%,30岁阳性率为0.54%。羊膜腔穿刺分析显示,21-三体、18-三体高风险诊断符合率为100%(11/11,2/2),13-三体高风险诊断符合率为80.00%(4/5)。NIPT诊断胎儿染色体非整倍体疾病的敏感度为100.00%(2 784/2 784),总特异度为94.44%(17/18),总假阳性率为0.03%(1/2 784),假阴性率为0.00%(0/0)。结论渝东北地区NIPT检测胎儿染色体非整倍体疾病的阳性检出率为0.72%,检出主要以21-三体为主,占61%,≥40岁高龄孕妇NIPT阳性率较其他年龄组明显升高,达2.97%。     

血型抗体效价在孕期检测的必要性

目的探讨孕期检测孕妇血型抗体效价的必要性。方法以产前检测均为O型血的98例孕妇作为研究对象,予以血型抗体效价检测。对98例新生儿出生后行血型血清学检查、溶血试验及胆红素检测,对孕妇血型抗体与新生儿溶血病(HDN)发病率的关系进行综合评判。结果孕妇血型抗体滴度与HDN发病率呈正相关(P0.05)。将父母血型不合的新生儿(母-父-婴)分为3组:O-A-A组、O-B-B组、O-AB-A/O-AB-B组,各组HDN发病率分别为45.5%、34.2%、33.3%,差异无统计学意义(P0.05)。结论孕期进行血型抗体效价的检测,可以及早发现母婴血型不合的问题,降低HDN发病率。     

HBV-DNA阳性孕妇产后脐带血及乳汁中HBV-DNA载量的检测分析

目的 通过荧光定量PCR(FQ-PCR)方法检测血清HBV-DNA阳性产妇产后脐带血及乳汁HBV-DNA载量,探讨胎儿宫内感染HBV及乳汁携带HBV与产妇HBV-DNA载量的相关性.方法 2010年1月至2012年11月,选取在产科住院分娩的HBV-DNA阳性产妇149例,采用FQ-PCR方法检测产妇产前血清、产后脐带血和乳汁HBV-DNA载量.结果 ①选择有HBV感染史产妇304例中149例HBV-DNA阳性,阳性率为49.01％(149/304);②149例HBV-DNA阳性产妇胎儿宫内感染为42.95％；HBV-DNA阳性孕妇乳汁中HBV-DNA阳性率(71.14％)显著高于脐血(42.95％)(P＜0.01); HBV-DNA≥1×105组脐带血、初乳及满月乳中HBV-DNA阳性率(61.29％、82.80％、100.00％)显著高于1×103≤HBV-DNA＜1 ×105组(12.50％、51.79％、75.00％)(P＜0.01)；满月乳中HBV-DNA阳性率(90.60％)显著高于初乳(71.14％)(P＜0.01).结论 HBV-DNA阳性孕妇胎儿HBV的宫内感染率及乳汁中携带HBV的阳性率高,与孕妇血清HBV-DNA载量正相关,阻断HBV的宫内感染及减少乳汁携带HBV者,必须有效的降低孕妇血中的HBV-DNA载量水平.     

广西南宁地区妊娠期巨细胞病毒感染情况与不良妊娠结局分析

目的了解广西壮族自治区南宁地区妊娠妇女巨细胞病毒(CMV)感染与不良妊娠结局之间的关系。方法采用酶联免疫吸附试验(ELISA)检测62 361例产前检查孕妇静脉血CMV-IgM;采用ELISA法检测11 086例产前诊断孕妇脐带血CMV-IgM,CMV-IgM阳性者采用聚合酶链反应(PCR)检测脐带血、羊水和绒毛CMV-DNA;依据临床不良妊娠情况进行分组,分为正常妊娠组和不良妊娠组(有不良妊娠和不良妊娠史者)。结果 (1)产前检查:孕妇静脉血CMV-IgM总阳性率为0.95%(594/62 361)。正常妊娠组CMV-IgM总阳性率为0.83%(467/56 533),其中早、中、晚孕期CMV-IgM阳性率分别为0.75%(228/30533)、0.91%(229/25204)和1.26%(10/796);不良妊娠组CMV-IgM总阳性率为2.18%(127/5 828),其中早、中、晚孕期CMV-IgM阳性率分别为1.31%(31/2 365)、2.67%(84/3 147)、3.80%(12/316)。不良妊娠组CMV-IgM总阳性率明显高于正常妊娠组(2.18%比0.83%,χ~2=102.525,P=0.000)。(2)产前诊断:孕妇CMV-DNA总阳性率为0.42%(47/11 086)。正常妊娠组CMV-DNA总阳性率为0.14%(5/3 687),脐带血、羊水、绒毛的CMV-DNA阳性率分别为0.14%(3/2 193)、0.15%(2/1 345)和0%(0/149);不良妊娠组CMV-DNA总阳性率为0.57%(42/7 399),脐带血、羊水和绒毛CMV-DNA阳性率分别为0.52%(25/4 851)、0.67%(16/2 392)、0.64%(1/156)。不良妊娠组CMV-DNA总阳性率明显高于正常妊娠组(0.57%比0.14%,χ~2=95.429,P=0.000)。结论南宁地区妊娠妇女CMV感染比例较高,存在一定母婴传播率,CMV感染与不良妊娠结局有关。     

荧光定量PCR技术在血液筛查中的应用及可行性分析

目的　了解 ELISA法筛查血液乙型肝炎病毒 ( hepatitis B virus,HBV)的漏检率 ,探讨荧光定量聚合酶链反应 ( Flurescence quantitative PCR,FQ-PCR)技术用于混合血浆标本病毒核酸检测的可行性。　方法　应用 FQ-PCR技术对常规 ELISA初复检正常献血者 (包括无偿献血者和个体献血者 )的微量血浆汇集池标本 ( 1 0人份× 2 0 μ1 )进行 HBV DNA检测 ,再对阳性汇集池中的标本进行单份检测。用双蒸水和 HBV DNA阴性汇集池中的血浆标本分别对 HBV DNA标准品(浓度为 1 0 3拷贝 /ml)作 1 0～ 5 0倍稀释后行 FQ-PCR测定 ,观察不同的血浆标本混合后是否存在 Taq酶抑制物的叠加作用及对 PCR结果有无影响。对浓度为 1 0拷贝 /ml～ 1 0 4拷贝 /ml的 HBV DNA标准品分别进行 FQ-PCR检测 ,确定试剂盒的敏感度。　结果　 1 2 0 0份无偿献血者标本中有 1 1例 HBV DNA阳性 ( 0 .92 % ) ;4 70份个体献血者标本中有 1 0例 HBVDNA阳性 ( 2 .1 3% )。双蒸水与混合血浆稀释的 HBV DNA标准品的 FQ-PCR检测结果 (定性 )完全一致。试剂盒的检出下限为 1 0 2拷贝 /ml。　结论　 ELISA法筛查血液存在较高的 HBV漏检率 ,FQ-PCR用于混合血浆标本的病毒核酸检测是可行的     

丙型肝炎患者血清HCVRNA含量与抗HCV和ALT的相关性

目的 探讨丙型肝炎病毒患者血清丙型肝炎病毒核酸（HCVRNA）、丙型肝炎病毒抗体（抗HCV）和丙氨酸氨基转移酶（ALT）的相关性。方法 取123例确诊丙型肝炎的患者的血清，采用荧光定量PER技术检测其HCVRNA含量，用ELISA法检测抗HCV和酶动力学法检测ALT浓度水平，并对所得数据进行统计学分析。结果 根据HCVRNA含量（拷贝／L）将资料分为〈10^5组、10^5-6组、10^7-8组和〉10^9组，各组的抗HCV阳性率分别为72．4％、84．6％、100％和100％；ALT均值（IU）分别为38．8、88．2、112．1和128．3；ALT异常率分别为24．1%、65．4％、88．9％和100％。HCVRNA含量对数值与ALT线性相关关系分析和显著性检验呈正相关（r=0．441，P〈0．05）。结论 抗HCV阳性率、ALT的异常率及平均值与HCVRNA的含量有关，HCVRNA含量越高，抗HCV阳性率和枷含量的均值与异常率越高；HCVRNA含量与ALT含量水平呈正相关。     

Fetal RHD genotyping by analysis of maternal plasma in a mixed population

Background: Maternal plasma analysis for the determination of the fetal RHD status is an exciting tool for the management of RhD‐negative pregnant women, specially sensitized women. We assessed the accuracy of fetal RHD genotyping by analysis of maternal plasma in a multi‐ethnic population. Methods: We analyzed plasma samples from 88 RhD‐negative pregnant women between 11 and 39 weeks of gestation, median age of 28 years old to determine the fetal RHD genotype. This population was from Southeastern Brazil with high mixed ethnic background. Fourteen patients (16%) had anti‐D alloantibody. We used Taqman primers and probes to detect by real‐time PCR, exons 4, 5, and 10 of RHD. As internal controls we used primers/probes sets to SRY and CCR5. Peripheral or umbilical cord bloods from respective nenonates were collected during delivery and hemagglutination was performed. Results: Fifty‐eight samples (66%) were genotyped as RHD+, 27 samples (31%) showed complete absence of RHD and 3 samples (3 %) presented a D variant (RHDψ). All the results agreed with the neonatal typing, including the three fetuses with the RHDψ, phenotyped as RhD‐negative. Thus, the accuracy of the fetal RHD genotyping in this mixed population was 100%. The earliest pregnancy in which fetal RHD was detected was 11 weeks. Conclusion: Our findings indicate that the accuracy of RHD gene using three regions (exons 4, 5, and 10) can be sufficient for clinical application in a multi‐ethnic population. This knowledge helped us on the development of a feasible protocol for fetal RHD genotyping on DNA from maternal plasma for our population. J. Clin. Lab. Anal. 25:100–104, 2011. © 2011 Wiley‐Liss, Inc.     

胎儿腹壁畸形的超声诊断

目的 探讨超声对胎儿腹壁畸形的诊断及鉴别诊断。方法 孕中期胎儿常规超声筛查,观察胎儿腹壁连续性,脐孔大小。首诊检查发现胎儿腹壁缺损6例,根据脐带位置确诊腹裂或脐膨出。结果 超声对胎儿腹壁缺损的诊断符合率为100％。结论超声对胎儿腹壁畸形的诊断孕龄越来越早。     

Laboratory Monitoring of Mother,Fetus, and Newborn in Hemolytic Disease of Fetus and Newborn

BackgroundLaboratory monitoring of mother, fetus, and newborn in hemolytic disease of fetus and newborn (HDFN) aims to guide clinicians and the immunized women to focus on the most serious problems of alloimmunization and thus minimize the consequences of HDFN in general and of anti-D in particular. Here, we present the current approach of laboratory screening and testing for prevention and monitoring of HDFN at the Copenhagen University Hospital in Denmark.SummaryAll pregnant women are typed and screened in the 1st trimester. This serves to identify the RhD-negative pregnant women who at gestational age (GA) of 25 weeks are offered a second screen test and a non-invasive fetal RhD prediction. At GA 29 weeks, and again after delivery, non-immunized RhD-negative women carrying an RhD-positive fetus are offered Rh immunoglobulin. If the 1st trimester screen reveals an alloantibody, antenatal investigation is initiated. This also includes RhD-positive women with alloantibodies. Specificity and titer are determined, the fetal phenotype is predicted by non-invasive genotyping based on cell-free DNA (RhD, K, Rhc, RhC, RhE, ABO), and serial monitoring of titer commences. Based on titers and specificity, monitoring with serial peak systolic velocity measurements in the fetal middle cerebral artery to detect anemia will take place. Intrauterine transfusion is given when fetal anemia is suspected. Monitoring of the newborn by titer and survival of fetal red blood cells by flow cytometry will help predict the length of the recovery of the newborn.     

First-trimester determination of fetal gender by ultrasound.

OBJECTIVE: To assess the accuracy of fetal sex determination at 11-14 weeks of gestation. METHODS: Fetal gender assessment by ultrasound was prospectively carried out in 172 singleton pregnancies at 11-14 weeks of gestation immediately before chorionic villus sampling for karyotyping. The genital region was examined in a midsagittal plane and the fetal gender was assigned as male if the angle of the genital tubercle to a horizontal line through the lumbosacral skin surface was greater than 30 degrees and female when the genital tubercle was parallel or convergent (less than 30 degrees) to the horizontal line. RESULTS: The accuracy of sex determination increased with gestation from 70.3% at 11 weeks, to 98.7% at 12 weeks and 100% at 13 weeks. In the male fetuses, there was a significant increase in the angle of the genital tubercle from the horizontal with crown-rump length. Male fetuses were wrongly assigned as female in 56% of cases at 11 weeks, 3% at 12 weeks and 0% at 13 weeks. In contrast, only 5% of the female fetuses at 11 weeks were incorrectly assigned as male and this false-positive rate was 0% at 12 and 13 weeks. CONCLUSION: The clinical value of determination of fetal sex by ultrasound is in deciding whether to carry out prenatal invasive testing in pregnancies at risk of sex-linked genetic abnormalities, because invasive testing would be necessary only in pregnancies with male fetuses. Our results suggest that a final decision on invasive testing for sex-linked conditions should be undertaken only after 12 weeks of gestation.     

Prediction of fetal D status from maternal plasma: introduction of a new noninvasive fetal RHD genotyping service

BACKGROUND: Invasive procedures to obtain fetal DNA for prenatal blood grouping present a risk to the fetus. During pregnancy, cell-free fetal DNA is present in maternal blood. The detection of RHD sequences in maternal plasma has been used to predict fetal D status, based on the assumption that RHD is absent in D- genomes. STUDY DESIGN AND METHODS: Real-time PCR assays were designed to distinguish RHD from RHDpsi (possessed by the majority of D- black Africans). Plasma-derived DNA from 137 D- women was subjected to real-time PCR to detect fetal RHD and Y chromosome-associated SRY sequences. The accuracy of RHD genotyping from maternal plasma was investigated by comparing results with those obtained by conventional RHD genotyping from fetal tissue or serologic tests on the infant's RBCs. The quantity of fetal DNA in maternal plasma was investigated in 94 pregnancies. RESULTS: Fetal D status was predicted with 100-percent accuracy from maternal plasma. The number of copies of fetal DNA in maternal plasma was found to increase with gestation. CONCLUSION: Combination of the sensitivity of real-time PCR with an improved RHD typing assay to distinguish RHD from RHDpsi enables highly accurate prediction of fetal D status from maternal plasma. This has resulted in the implementation of a clinical noninvasive fetal RHD genotyping service.     

妊娠23~24周三维容积超声预测晚发型胎儿生长受限的应用价值

目的探讨妊娠23~24周三维容积超声部分肢体体积预测晚发型胎儿生长受限(FGR)的应用价值。方法选取在我院行产前检查并于出生后最终确诊为晚发型FGR的产妇74例(病例组),另选同期正常产妇200例为对照组。应用超声测量妊娠23~24周胎儿二维超声参数:双顶径、头围、腹围、股骨长度,记录二维超声参数生成的胎儿体质量(EFW1);应有三维容积超声测量胎儿上臂中段50%的体积(AVol)和大腿中段50%的体积(TVol),EFW1联合AVol或TVol生成EFW2或EFW3。比较两组上述各参数差异;应用受试者工作特征(ROC)曲线分析各个参数预测晚发型FGR的价值。结果两组双顶径、头围、腹围、股骨长度、EFW1、EFW2、EFW3比较差异均无统计学意义;两组AVol和TVol比较差异均有统计学意义(均P<0.05)。ROC曲线分析显示,AVol和TVol截断值分别为4.5 ml和9.4 ml,预测晚发型FGR的敏感性、特异性、准确率、曲线下面积分别为63.5%vs.69.4%、89.4%vs.88.1%、81.4%vs.83.2%、0.70 vs.0.74;二者曲线下面积比较差异无统计学意义;二者联合预测晚发型FGR的敏感性、特异性及准确率分别为79.0%、94.8%及90.1%,均较二者单独应用的诊断效能高。结论妊娠23~24周胎儿的AVol和TVol可作为预测晚发型FGR的特征性指标。     

四腔心切面联合三血管气管切面彩色多普勒超声在孕11~13+6周胎儿严重先天性心脏畸形筛查中的应用价值

目的探讨四腔心切面联合三血管气管切面彩色多普勒超声在孕11~13⁺⁶周胎儿严重先天性心脏畸形筛查中的应用价值。 方法选择2018年1月至12月在四川省妇幼保健院行孕期检查的9756例孕妇,分别于孕 11~13⁺⁶周及孕16~24周进行胎儿心脏超声检查。采用四腔心切面联合三血管气管切面彩色多普勒超声对孕11~13⁺⁶周胎儿筛查心脏畸形,采用标准化胎儿超声心动图筛查孕16~24周胎儿心脏畸形,并对分娩后所有新生儿及引产胎儿进行随访。 结果9756例胎儿孕11~13⁺⁶周超声筛查发现心脏畸形38例（51.4%,38/74）,其中非严重心脏畸形5例（14.2%,5/35）,严重心脏畸形33例（84.6%,33/39）。出生或引产后诊断先天性心脏畸形74例,其中严重先天性心脏畸形39例,非严重心脏畸形35例。 结论运用四腔心切面联合三血管气管切面彩色多普勒超声能筛查出大部分严重胎儿心脏畸形,可为临床咨询、预后提供及时有力的证据。     

胎儿超声心动图检查定量11~17周正常胎儿主动脉和肺动脉内径Z-评分的初步研究

目的探讨早孕期（11^＋0周~13^＋6周）和中孕早期（14^＋0周~17^＋6周）胎儿主动脉内径（AO）与肺动脉内径（PA）与胎儿生物学生长参数的相关性,初步建立早孕期和中孕早期胎儿AO与PA的正常参考值范围及Z-评分方程,并评价方程的有效性。 方法随机选取孕周（GA）为（11^＋0周~17^＋6周）正常单胎胎儿270例,将成功显示左、右心室流出道切面的245例胎儿纳入研究,获得胎儿顶臀径（CRL）、双顶径（BPD）、股骨长径（FL）、孕周（GA）等生物学生长参数。在胎儿左、右心室流出道切面测量收缩末期AO及PA,以GA、BPD和FL作为独立自变量,AO及PA作为因变量,建立AO、PA的正常参考值范围,并对每个参数的绝对残差（SD）进行加权回归,建立Z-评分方程。 结果采用简单的线性回归模型,可以很好地描述AO和PA与非心脏生物特征参数（BPD、FL、GA）的关系。AO、PA与GA、BPD、FL均呈显著线性相关（GA与AO：r=0.9276,GA与PA：r=0.9271,BPD与AO：r=0.9551,BPD与PA：r=0.9558,FL与AO：r=0.9462,FL与PA：r=0.9483,均<0.001）,其中与BPD的相关性最强。 结论正常早孕期及中孕早期胎儿的AO、PA随着孕周的增加而增长,本研究初步建立了正常胎儿早孕期及中孕早期AO、PA的参考范围及其Z-评分方程。为早孕期及中孕早期评估胎儿大血管生长提供精确的参考标准,在早期筛查或诊断胎儿先天性心脏畸形方面具有潜在的应用价值。