引物延伸预扩增后STR分型在鉴定母血中胎儿有核红细胞的应用

目的 探讨一种鉴定母血中胎儿有核红细胞（NRBC）的方法，为无创性产前基因诊断创造必要条件。方法 联苯胺染色识别孕妇外周血中NRBC，经显微操作获取，并以引物延伸预扩增（PEP）对单个NRBC进行全基凶组扩增后，用短串联重复序列（STR）分析其基因型，与父母基因型比对，确定该细胞的来源。结果 28例轻型β地中海贫血孕妇外周血样本中每例发现NRBC4～13个／5ml，经鉴定每例有胎儿NRBC2～8个／5ml，约43．6％的NRBC来源于胎儿。结论 PEP后STR基因型分析能有效鉴定孕妇外周血中NRBC的来源，使应用单个胎儿NRBC进行产前基因诊断成为可能。     

Isolation of fetal nucleated red blood cells from maternal blood in normal and aneuploid pregnancies.

Fetal nucleated red blood cells (NRBC) have been widely reported in maternal blood during pregnancy. However, there is no consensus with regard to their presence in all pregnancies. Therefore, the usefulness of developing a fetal NRBC-based noninvasive method suitable for clinical prenatal diagnosis remains uncertain. Fluorescence in situ hybridization (FISH) method was used to evaluate the ability of one of our own monoclonal antibodies (mAb), 2B7.4, to isolate fetal NRBC from maternal blood by magnetic activated cell sorting (MACS). Our mAb was able to isolate from 25 to 822 NRBC from all of the 45 maternal blood samples included in this study. A correct diagnosis was achieved in 21 out of 24 pregnancies carrying trisomic fetuses (87.5%), with a fetal/maternal NRBC frequency of 8.4%. In contrast, a significantly lower percentage of fetal NRBC (0.2%) was observed in 22% of pregnancies carrying a chromosomally normal male fetus, that were correctly predicted. In conclusion, using 2B7.4 mAb we succeeded in isolating NRBC from the maternal blood samples, but most of the isolated cells were maternal in origin. Nevertheless, a higher number of fetal NRBC was found in the peripheral blood of pregnant women carrying aneuploid fetuses, which could allow development of a screening method for prenatal diagnosis of fetal aneuploidies.     

母血中胎儿有核红细胞的分离及无创性产前基因诊断研究

目的 从母血循环中分离、富集、鉴定和提取胎儿有核红细胞,建立遗传病无创性产前基因诊断方法。方法 取２５名妊娠８～３６周的孕妇静脉血,加载在细胞分离液Ｈｉｓｔｏｐａｑｕｅ１．０７７上分离单个核细胞（ＭＮＣ）;用包被ＣＤ７１抗体的ＤｙｎａｂｅａｄｓＭ－４５０ＣＤ２１免疫磁珠或包被ＣＤ４５抗体的ＤｙｎａｂｅａｄｓＭ－４５０ＣＤ４５免疫磁珠富集ＭＮＣ中的胎儿有核红细胞：ａｎｔｉ－ξ－ｂｉｏｔｉｎ或ａｎｔｉ     

应用密度梯度离心结合免疫磁珠法富集孕妇外周血中胎儿NRBC的实验研究

目的研究利用不连续密度梯度离心结合免疫磁珠方法,分选孕妇外周血中的胎儿有核红细胞(NRBC)。方法采集21名孕周在12-28周的孕妇外周血10ml,经不连续密度梯度离心后,再用免疫磁珠法分选和富集胎儿NRBC,同时对富集前后的孕妇外周血进行瑞氏染色和胎儿血红蛋白(fetal hemoglobin,HbF)特异性抗体标记、识别。结果未经分选、富集,21名孕妇外周血有核细胞层涂片经瑞氏染色检查均未发现NRBC;梯度离心富集后21名孕妇中有11例外周血有核细胞涂片找到1～8个NRBC;再经免疫磁珠分离后有12例涂片发现1～7个NRBC,同时进行HbF特异性抗体标记,证实其NRBC阳性涂片中均存在胎源性NRBC。结论本研究初步建立了分选、富集胎儿NRBC的实验方法,通过进一步的方法学改进,有望用于产前基因筛查的研究。     

应用密度梯度离心结合免疫磁珠法富集孕妇外周血中胎儿NRBC的实验研究

目的 研究利用不连续密度梯度离心结合免疫磁珠方法,分选孕妇外周血中的胎儿有核红细胞(NRBC).方法 采集21名孕周在12-28周的孕妇外周血10ml,经不连续密度梯度离心后,再用免疫磁珠法分选和富集胎儿NRBC,同时对富集前后的孕妇外周血进行瑞氏染色和胎儿血红蛋白(fetal hemoglobin,HbF)特异性抗体标记、识别.结果 未经分选、富集,21名孕妇外周血有核细胞层涂片经瑞氏染色检查均未发现NRBC;梯度离心富集后21名孕妇中有11例外周血有核细胞涂片找到1～8个NRBC;再经免疫磁珠分离后有12例涂片发现1～7个NRBC,同时进行HbF特异性抗体标记,证实其NRBC阳性涂片中均存在胎源性NRBC.结论 本研究初步建立了分选、富集胎儿NRBC的实验方法,通过进一步的方法学改进,有望用于产前基因筛查的研究.     

Determination of sibship by PCR-amplified short tandem repeat analysis in Taiwan

BACKGROUND: Sibship determination for any two persons whose parents have died is one of the most fundamental issues of personal identification, second only to those of a parent-child relationship. STUDY DESIGN AND METHODS: By automated fluorescence analysis of a PCR-amplified short tandem repeat (STR) system in conjunction with capillary electrophoresis, a panel of up to 15 polymorphic, autosomal, unlinked STR loci was used to investigate sibship index (SI) values in a cohort of 126 true sibling pairs. These SI values were then compared with those of 126 random pairs. RESULTS: The 15-loci STR panel provides a cumulative power of exclusion of 0. 9999997. Of the 126 random pairs, 124 (98.4%) had cumulative sibship indices (CSIs) of <1.0, and none had a CSI of >3.0 (median, 0.0101; range, 0.0000003-2.5376). In contrast, 107 (85%) of the 126 sibling pairs had a CSI of >100 (median, 5,579.9853; range, 0.0747-9,406,829, 249.8461). However, five pairs (4%) of the sibling group had a CSI of <3.0. True sibship was confirmed for this particular group by additional paternity testing and mitochondrial DNA sequence analysis. Among a total of 1890 observations (15 loci x 126 pairs), two alleles per locus were shared 760 times (40.21%) (mean, 6.03 loci; range, 1-10) in the sibling group, but only 192 times (10.16%) in the random group (mean, 1.52 loci; range, 1-5) (p<0.001). No alleles were shared 696 times (36.83%) in the unrelated pairs, as compared to 176 times (9.31%) in the sibling group (p<0.001). A polarized distribution was not noted in the sharing of single alleles in either the random or the sibling group: 1002 observations (53.02%) and 954 observations (50.48%), respectively. CONCLUSION: Highly polymorphic STR analysis can be discriminative in most sibship determinations, and the sharing of two alleles per locus is most informative in indicating sibship. Complementary mitochondrial DNA sequence analysis is mandatory in a few cases to exclude or establish true sibship when CSIs are equivocal and neither parent is available.     

Diagnosis of transfusion-associated graft-vs.-host disease: the importance of short tandem repeat analysis

Transfusion-associated graft-vs.-host disease (TA-GvHD) can occur following transfusion of blood products containing immunocompetent lymphocytes, usually from HLA homozygous donors, into immunocompromised patients sharing one HLA haplotype with the donor. The diagnosis of TA-GvHD may be delayed due to the initial nonspecific clinical features involved. Investigations to detect the presence of donor-derived cells in the blood and/or affected tissues of the recipient are essential to confirm the diagnosis. We report the investigation of suspected TA-GvHD using short tandem repeat (STR) analysis, to detect the presence of donor cells (chimerism), in an immunocompetent patient admitted for coronary artery bypass surgery. Peripheral blood and skin biopsies (from affected and nonaffected sites) from the patient and peripheral blood samples from the implicated donors were taken for HLA typing and STR analysis. STR analysis revealed the presence of donor material in the patient's peripheral blood sample and in DNA extracted from the affected skin biopsy but not the unaffected biopsy, suggesting lymphocytes from this donor were responsible for the development of TA-GvHD. Furthermore, HLA typing results supported the diagnosis of TA-GvHD. These data demonstrate the use of STR and HLA analysis as effective tools in the diagnosis of TA-GvHD.     

Resolution of a serum sample mix-up through the use of short tandem repeat DNA typing

BACKGROUND: A sample mix-up occurred in a tissue procurement laboratory in which aliquots of serum from two tissue donors were accidentally mislabeled. The clues to the apparent mixup involved discrepant Hepatitis C test results. In an attempt to resolve the apparent mix up, DNA typing was performed using serum samples as a possible source of genomic DNA. STUDY DESIGN AND METHODS: Two hundred microliter aliquots of two reference sera and aliquots prepared from them were subjected to DNA extraction. PCR amplification of 9 STR loci was performed on the extracts and amplicons were analyzed by capillary electrophoresis. RESULTS: About 1 microg/ml of DNA was recovered from all serum samples and was of sufficient quality to direct the amplification of most, if not all STR loci allowing the mislabeled specimens to be traced to the proper tissue donor. CONCLUSIONS: Serum is a useful source of genomic DNA for STR analysis in situations in which such samples are the only source of DNA for testing. Interestingly, one of the tissue donors on life support and repeatedly receiving blood products, exhibited a mixed DNA profile indicative of the presence of DNA from multiple individuals in the bloodstream.     

短串联重复序列监测异基因造血干细胞移植后的植入状态及其演变过程

目的：监测和观察异基因造血干细胞移植后的植入状态及其演变过程。方法：于2003—01／2004—12在中国造血干细胞库深圳分库采集完成人类白细胞抗原配型的6对供受者标本。采用荧光标记复合扩增短串联重复序列检测技术，对6对供受者标本移植前后的系列血样进行DSS1179，1321S11，D18S51，D5S818，D13S317，D7S820，D3S1358，vWA，FGA共9个短串联重复序列位点和Amelogenin性别位点的检测，找出供受者间的差异基因，通过移植后不同时间段患者基因型的转变情况，判断供者细胞是否植入以及嵌合体类型。结果：供受者6对样本均获得满意的9个短串联重复序列位点和1个性别位点的分型结果。6对移植标本短串联重复序列差异基因由受者型向供者型转化的时间为14～23d。6对样本间存在明显差异。第3对、第5对在第14天已为供者完全嵌合状态，而第1对在第16天、第4对在第18天尚为供受者混合嵌合体。但6对样本都是一个从混合嵌合向完全嵌合发展的良好趋势。结论：①6对移植标本短串联重复序列差异基因由受者型向供者型转化的时间为14～23d，6对样本都有从混合嵌合向完全嵌合发展的良好趋势。②采用荧光标记复合扩增短串联重复序列方法可精确地测量聚合酶链反应产物的数量，描述造血干细胞的植入程度及植入的整个演变过程。     

中国朝鲜族人群6个短串联重复序列位点的遗传多态性

背景：许多学者对不同国家、地区、民族人群的短串联重复序列多态性进行了研究报道,但朝鲜族人群短串联重复序列位点的多态性如何？目的：了解中国朝鲜族人群D16S539,D7S820,D13S317,CSF1PO,TPOX,THO1 6个短串联重复序列位点的遗传多态性分布,获得相应多态位点的群体遗传学数据.设计：单一样本观察.单位：牡丹江医学院生物教研室和DNA分析检测室.材料：收集2001-01/02牡丹江地区无血缘关系的朝鲜族个体100份血样.方法：应用聚合酶链反应扩增片段长度多态性分析方法对100名无血缘关系的朝鲜族个体进行样本基因型检测.主要观察指标：D16S539,D7S820,D13S317,CSF1PO,TPOX,THO1 6个短串联重复序列位点的样本基因型.结果：①D16S539基因座,观察到6个等位基因,18种基因型.②D7S820基因座,观察到7个等位基因,22种基因型.③D13S317基因座,观察到7个等位基因,23种基因型.④CSF1PO基因座,观察到6个等位基因,16种基因型.⑤TPOX基因座,观察到6个等位基因,11种基因型.⑥THO1基因座,观察到5个等位基因,12种基因型.结论：6个位点等位片段多态性分布均符合Hardy-Weinberg平衡定律.且具有较高的杂合度,所得到的等位基因频率等数据可以为中国朝鲜族人群遗传学研究提供依据.     

Use of X-linked short tandem repeat loci in routine parentage casework

BACKGROUND: Dealing with genetic inconsistencies in parentage testing, especially in motherless cases, remains a continual difficulty. STUDY DESIGN AND METHODS: Four difficult cases, comprising two trios and two duos, were selected from routine parentage testing casework. In these, relatively low combined paternity indices were observed as a result of few discrepant loci that were treated as being due to paternal mutations. An additional eight short tandem repeat (STR) loci along the X chromosome were studied in the alleged father and female child to try and help resolve these cases. RESULTS: In all four cases, the X chromosome haplotypes in the alleged father were different from those in the child, showing decisively that the alleged father could be excluded from being the biologic father of the child. CONCLUSION: In recent times the study of X chromosome haplotypes has been shown to be useful in parentage testing where the alleged father is absent and where only his close relatives are available for testing. This work demonstrates that such studies can also prove valuable in the testing of standard trios and duos in cases where there only a few genetic inconsistencies amongst the loci tested, making it difficult to distinguish between paternal mutations and a close relative of the alleged father being the biologic father.     

短串联重复序列监测异基因造血干细胞移植后的植入状态及其演变过程

目的:监测和观察异基因造血干细胞移植后的植入状态及其演变过程。方法:于2003-01/2004-12在中国造血干细胞库深圳分库采集完成人类白细胞抗原配型的6对供受者标本。采用荧光标记复合扩增短串联重复序列检测技术,对6对供受者标本移植前后的系列血样进行D8S1179,D21S11,D18S51,D5S818,D13S317,D7S820,D3S1358,vWA,FGA共9个短串联重复序列位点和Amelogenin性别位点的检测,找出供受者间的差异基因,通过移植后不同时间段患者基因型的转变情况,判断供者细胞是否植入以及嵌合体类型。结果:供受者6对样本均获得满意的9个短串联重复序列位点和1个性别位点的分型结果。6对移植标本短串联重复序列差异基因由受者型向供者型转化的时间为14～23d。6对样本间存在明显差异。第3对、第5对在第14天已为供者完全嵌合状态,而第1对在第16天、第4对在第18天尚为供受者混合嵌合体。但6对样本都是一个从混合嵌合向完全嵌合发展的良好趋势。结论:①6对移植标本短串联重复序列差异基因由受者型向供者型转化的时间为14～23d,6对样本都有从混合嵌合向完全嵌合发展的良好趋势。②采用荧光标记复合扩增短串联重复序列方法可精确地测量聚合酶链反应产物的数量,描述造血干细胞的植入程度及植入的整个演变过程。     

中国朝鲜族人群6个短串联重复序列位点的遗传多态性

背景许多学者对不同国家、地区、民族人群的短串联重复序列多态性进行了研究报道,但朝鲜族人群短串联重复序列位点的多态性如何?目的了解中国朝鲜族人群D16S539,D7S820,D13S317,CSF1PO,TPOX,THO1 6个短串联重复序列位点的遗传多态性分布,获得相应多态位点的群体遗传学数据.设计单一样本观察.单位牡丹江医学院生物教研室和DNA分析检测室.材料收集2001-01/02牡丹江地区无血缘关系的朝鲜族个体100份血样.方法应用聚合酶链反应扩增片段长度多态性分析方法对100名无血缘关系的朝鲜族个体进行样本基因型检测.主要观察指标D16S539,D7S820,D13S317,CSF1PO,TPOX,THO1 6个短串联重复序列位点的样本基因型.结果①D16S539基因座,观察到6个等位基因,18种基因型.②D7S820基因座,观察到7个等位基因,22种基因型.③D13S317基因座,观察到7个等位基因,23种基因型.④CSF1PO基因座,观察到6个等位基因,16种基因型.⑤TPOX基因座,观察到6个等位基因,11种基因型.⑥THO1基因座,观察到5个等位基因,12种基因型.结论6个位点等位片段多态性分布均符合Hardy-Weinberg平衡定律.且具有较高的杂合度,所得到的等位基因频率等数据可以为中国朝鲜族人群遗传学研究提供依据.     

Enrichment of rare fetal cells from maternal peripheral blood

The use of fetal cells enriched from maternal peripheral blood for genetic analysis would be a significant advancement for prenatal diagnosis. For over two decades, researchers have employed different techniques (including density gradients, red blood cell lysis, fluorescence-activated cell sorter and magnetic cell separation) to enrich fetal cells that exist in low levels in maternal blood. Despite extensive attempts by various groups and companies, reliable isolation of a sufficient amount of fetal nucleated cells remains a monumental challenge. AVIVA Biosciences Corp. has been developing a technology that allows significant enrichment and recovery of rare fetal cells from maternal peripheral blood. This review will characterize fetal cell types present in maternal blood, technologies used to enrich for these cells and the issues involved with rare fetal cell separation.     

Screening for trisomy 21 by fetal nuchal translucency and maternal age: a multicenter project in Germany, Austria and Switzerland.

OBJECTIVE: To examine the effectiveness of screening for trisomy 21 by a combination of maternal age and fetal nuchal translucency thickness at 10-14 weeks of gestation in Germany, Austria and Switzerland. METHODS: This was a multicenter study of screening for trisomy 21 by a combination of maternal age and fetal nuchal translucency thickness at 10-14 weeks of gestation. All the sonographers involved in the study had received The Fetal Medicine Foundation Certificate of Competence in the 10-14-week scan. Fetal nuchal translucency thickness and crown-rump length were measured in 23 805 singleton pregnancies with live fetuses. In each case the risk for trisomy 21 was estimated on the basis of maternal age and fetal nuchal translucency thickness for crown-rump length with the use of The Fetal Medicine Foundation's software. The distribution of estimated risk was determined and the sensitivity and false-positive rate for a risk cut-off of 1 in 300 was calculated. RESULTS: Fetal nuchal translucency thickness was successfully measured in all 23 805 pregnancies and outcome was available in 21 959. The median maternal age was 33 (range 15-49) years and in 7935 (36.1%) the age was 35 years or greater. The median gestation at screening was 12 (10-14) weeks and the median fetal crown-rump length was 61 (range 38-84) mm. The estimated risk for trisomy 21 based on maternal age and fetal nuchal translucency thickness for crown-rump length was 1 in 300 or greater in 13.0% (2800 of 21 475) normal pregnancies, in 87.6% (184 of 210) of those with trisomy 21 and in 87.2% (239 of 274) with other chromosomal defects. CONCLUSIONS: In Germany, Austria and Switzerland the results of screening for chromosomal defects by measurement of fetal nuchal translucency thickness, in centers with appropriately qualified sonographers and using The Fetal Medicine Foundation's software, are similar to those reported in the UK using the same methodology.