Dielectrophoresis Activated Cell Sorting for Recovery of Fetal Erythroblasts

描述（由申请人提供）：目前的产前诊断方法本质上是侵入性的，并且有导致流产的重大风险；因此，并不是所有的孕妇都要做。因此，大量的胎儿遗传异常，如唐氏综合症，被遗漏。可靠的无创产前诊断方法长期以来一直在围产期医学中寻求。胎儿有核红细胞（nrbc）在母体血液中的存在是公认的，它们目前被认为是无创产前筛查和诊断的最佳目标。然而，这些细胞的检测仍然存在问题，因为它们在母体循环中的丰度很低，并且存在来自母体的类似nrbc。目前鉴定胎儿nrbc的方法依赖于机械分离方法，如流动激活细胞分选（FACS）或磁激活细胞分选（MACS），随后的细胞形态学分析，荧光原位杂交（FISH）或基于分子或遗传生物标志物的pcr分析。然而，根据国家儿童健康和人类发育研究所胎儿细胞分离研究（NIFTY），这些方法都没有显示出从母体血液中获得胎儿细胞的足够可靠性，用于常规产前诊断。因此，开发一种从母体血液中分离胎儿nrbc的有效方法是必要的，以便能够对少量这些细胞进行常规检测，以进行无创产前诊断。我们假设胎儿nrbc的独特形态及其独特的介电性质有助于通过介电电泳分离这些细胞。该提案描述了一个集成的设计和开发计划，导致一种新的小型化Dielectrophoresis Activated Cell Sorting （DACS）系统，用于从母体外周血中恢复胎儿nrbc。用DACS系统分离胎儿nrbc将使染色体和遗传异常的准确、早期诊断和筛查成为可能，而不会有流产的风险。此外，DACS系统可用于从外周血中分离其他形态上不同的细胞群，例如来自肿瘤或肿瘤前病变的细胞群。公共卫生相关性：DACS技术的成功开发将导致一种高度敏感和特异性的方法，用于无创识别和分离胎儿nrbc，允许及时识别胎儿的染色体和遗传异常，而没有标准程序相关的风险。一些确定的产前状况可以得到治疗，以改善未出生婴儿的前景。例如，生物素依赖症和甲基丙二酸血症这两种威胁生命的遗传性疾病，已经通过羊膜穿刺术诊断出来，并在子宫内进行治疗，从而生下了健康的婴儿。当胎儿的状况尚无法进行产前治疗时，产前诊断可以让父母做好心理准备，并计划最安全的时间、医院设施和分娩方法。

DESCRIPTION (provided by applicant): Current prenatal diagnostic methods are invasive in nature and carry significant risks of causing a miscarriage; therefore, they are not performed on all pregnant women. As a result, a large number of fetal genetic abnormalities, such as Down syndrome, are missed. Reliable methods of noninvasive prenatal diagnosis have long been sought in perinatal medicine. The presence of fetal nucleated red blood cells (NRBCs) in the maternal blood is well-established, and they are currently considered the best target for noninvasive prenatal screening and diagnosis. However, the detection of these cells remains problematic due to their low abundance in maternal circulation, and the presence of similar NRBCs that are of maternal origin. Current approaches for identification of fetal NRBCs rely on mechanical separation methods, such as flow-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS), subsequent analysis of cellular morphology, and fluorescence in situ hybridization (FISH)- or PCR-based analyses of molecular or genetic biomarkers. However, according to the National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) none of these methods have been shown to obtain fetal cells from maternal blood with sufficient reliability for routine prenatal diagnosis. Thus, the development of an efficient method for separating fetal NRBCs from maternal blood is necessary to enable the routine detection of a small numbers of these cells for noninvasive prenatal diagnosis. We hypothesize that the unique morphology of fetal NRBCs, and subsequently their unique dielectric properties, can facilitate separation of these cells by dielectrophoresis. This proposal describes an integrated design and development plan leading to a novel miniaturized Dielectrophoresis Activated Cell Sorting (DACS) system for recovery of fetal NRBCs from maternal peripheral blood. Isolation of fetal NRBCs with the DACS system will enable accurate, early diagnosis and screening for chromosomal and genetic abnormalities without risk of miscarriage. In addition, the DACS system can be adapted to separate other types of morphologically distinct cell populations from peripheral blood, such as those from a tumor or a pre-neoplastic lesion. PUBLIC HEALTH RELEVANCE: Successful development of the DACS technology will result in a highly sensitive and specific method for noninvasive identification and isolation of fetal NRBCs, allowing timely identification of chromosomal and genetic abnormalities in the fetus without the risks associated with standard procedures. Some of the identified prenatal conditions can then be treated to improve the outlook for the unborn baby. For example, biotin dependence and methylmalonic acidemia, both life-threatening inherited disorders, have been diagnosed by amniocentesis and treated in the womb, resulting in the births of healthy babies. When a fetus has a condition for which prenatal treatment is not yet possible, prenatal diagnosis permits parents to prepare themselves emotionally, and to plan the safest timing, hospital facility, and method of delivery.