FETAL ERYTHROID CELLS IN MATERNAL CIRCULATION

分子遗传学的最新进展使人们可以考虑 通过少量胎儿红细胞对胎儿进行基因检测 穿过胎盘进入母体循环的细胞 孕期 这样的母亲血液测试最终可能 补充，甚至取代这种侵入性的程序， 羊膜穿刺术和绒毛取样。 这对 这些分子技术的发展，首先明确确定 区分胎儿细胞和母体细胞， 胎儿红系细胞出现的妊娠变异性， 包括有核成红细胞。 我们有 开发了一种流式细胞术方法， 从母体中鉴定、定量和分离胎儿细胞 外周血样本。 我们的技术利用了 细胞表面红细胞谱系标志物血型糖蛋白A也是 多态性MN血型的遗传决定子，并利用 等位基因特异性单克隆抗体的M和N的形式， 血型糖蛋白A 通过用这两种抗体进行双标记， 使用双色流式细胞术，我们可以明确区分胎儿 在母亲的血液中，有50%的病例， 和孩子在这个基因座上是不一致的。 试点研究 我们已经确定，有可检测到的胎儿红系细胞， 绒毛取样前的母体循环（9-12周 生完孩子后，就有了一个完整的人生。 在这段时间内胎儿细胞频率的增加。我们建议申请 将这些技术应用于100对信息丰富的母体/胎儿对的群体 在正常交付后进行鉴定，并在整个过程中储存样品 怀孕 这些技术将被应用于事件 母亲/胎儿对有各种不良后果，包括产前 子痫这项研究还将为发展和 胎儿细胞富集和直接分选技术的应用 碘化丙啶阳性胎儿细胞（网织红细胞和有核细胞） 成红细胞）。 假定的胎儿细胞将作为群体被捕获 在试管中，或放置在载玻片网格上， 分析有核细胞。 排序的胎儿派生 网织红细胞和有核细胞将使用等位基因特异性 通过PCR检测血型糖蛋白A mRNA，通过PCR和/或FISH分析检测 在男性受孕体中存在Y染色体。

Recent advances in molecular genetics make it feasible to consider genetic testing of the fetus via the small numbers of fetal erythroid cells that migrate across the placenta into the maternal circulation during pregnancy. Such a maternal blood test could ultimately complement, or perhaps even replace such invasive procedures as amniocentesis and chorionic villus sampling. It is critical to the development of these molecular techniques to first definitively identify and distinguish fetal cells from maternal cells, and second determine the gestational variability in the occurrence of fetal erythroid cells, including nucleated erythroblasts, in the maternal circulation. We have developed a flow cytometric method of unambiguously and directly identifying, quantifying and isolating fetal cells from maternal peripheral blood samples. Our technique takes advantage of the fact that the cell surface erythroid lineage marker glycophorin A is also the genetic determinant of the polymorphic MN blood group, and utilizes allele-specific monoclonal antibodies to the M and N forms of the glycophorin A protein. By double labeling with these two antibodies and using two-color flow cytometry, we can unambiguously distinguish fetal cells in maternal blood in those 50 percent of cases where the mother and child are genetically discordant at this locus. In pilot studies we have determined that there are detectable fetal erythroid cells in the maternal circulation prior to chorionic villus sampling (9-12 weeks gestation) and after full-term uncomplicated birth, and that there is an increase in fetal cell frequency over this time. We propose to apply these techniques to a population of 100 informative maternal/fetal pairs identified following normal delivery with samples banked throughout gestation. These techniques will then be applied to incident maternal/fetal pairs with a variety of adverse outcomes, including pre- eclampsia. This study will also provide samples for the development and application of techniques for fetal cell enrichment and direct sorting of propidium iodide-positive fetal cells (reticulocytes and nucleated erythroblasts). Putative fetal cells will be captured as a population in a test tube, or laid out on a slide grid for direct microscopic analysis for nucleated cells. The fetal derivation of sorted reticulocytes and nucleated cells will be verified using allele-specific PCR for glycophorin A mRNA and by PCR and/or FISH analysis for the presence of a Y chromosome in male conceptuses.