HLA-G positive trophoblastic cells in transcervical samples and their isolation and analysis by laser microdissection and QF-PCR
HLA-G positive trophoblastic cells in transcervical samples and their isolation and analysis by laser microdissection and QF-PCR
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DOI：
10.1002/pd.511
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发表时间：
2003-01-01
影响因子：
3
通讯作者：
Adinolfi, M
中科院分区：
文献类型：
--
作者：
Bulmer, JN;Cioni, R;Adinolfi, M
目的应用特异性抗HLA-G单克隆抗体（G233）检测妊娠早期宫颈管标本中细胞滋养层细胞的频率。从一组四个选择的样品中，通过激光显微切割收集通过免疫染色鉴定的细胞，然后通过定量荧光聚合酶链反应（QF-PCR）测试父系来源的DNA标记物的存在，方法用抗HLA-233单克隆抗体G233对23例经宫颈移植的子宫内膜标本中的合胞体片段和细胞滋养层细胞进行免疫组化鉴定。G抗原和抗细胞角蛋白抗体。来自相同样品的载玻片也通过荧光原位杂交（FISH）进行检测，而选择的样品通过QF-PCR进行分析。通过激光显微切割收集合胞体片段和母体上皮细胞，并通过QF-结果所有男性胎儿的母亲宫颈标本经FISH检测均含有Y染色体特异性信号。用抗HLA-G单克隆抗体检测，约50%的标本中可检出细胞滋养层细胞成分，其中4例经免疫染色鉴定为细胞滋养层细胞或合胞体片段，用QF-PCR检测胎儿DNA标记，结果表明细胞滋养层细胞成分为胎儿来源。胎儿细胞在子宫下腔释放，可以分离和分析，用于单基因缺陷和非整倍体的产前诊断。版权所有（C）2002约翰威利父子有限公司
Objective To assess the frequency of cytotrophoblastic cells in endocervical samples collected by lavage at early stages of gestation using a specific anti-HLA-G McAb (G233). From a set of four selected samples, cells identified by immunostaining were collected by laser microdissection and then tested by quantitative fluorescent polymerase chain reaction (QF-PCR) for the presence of paternally derived DNA markers, in order to establish their fetal origin.Methods Syncytial fragments and cytotrophoblastic cells from 23 transcervical samples were identified by immunostaining with McAb G233 reacting against HLA-G antigen and with antibodies against cytokeratin. Slides from the same samples were also tested by fluorescent in situ hybridization (FISH), while selected samples were analysed by QF-PCR.Slides from four samples retrieved from mothers with male fetuses were immunolabelled and then cytotrophoblastic cells, syncytial fragments and maternal epithelial cells were collected by laser microdissection and tested by QF-PCR.Results All endocervical samples retrieved from mothers with male fetuses were found to contain some cells with chromosome Y-specific signals when tested by FISH. Using McAb anti- HLA-G, cytotrophoblastic cellular elements were detected in about 50% of the samples.From four samples, cellular elements identified by immunostaining as cytotrophoblast or syncytial fragments were collected by laser microdissection and shown to be of fetal origin when tested by QF-PCR for the presence of fetal DNA markers.Conclusions These results confirm that, during an early phase of gestation, fetal cells are released in the lower uterine cavity and that they can be isolated and analysed for prenatal diagnosis of single gene defects and aneuploidies. Copyright (C) 2002 John Wiley Sons, Ltd.