Dielectrophoresis Activated Cell Sorting for Recovery of Fetal Erythroblasts

说明(申请人提供)：目前的产前诊断方法本质上是侵入性的，具有导致流产的重大风险；因此，它们并不适用于所有孕妇。因此，唐氏综合症等大量胎儿基因异常被遗漏。围产期医学长期以来一直在寻求可靠的无创性产前诊断方法。胎儿有核红细胞(NRBCs)在母体血液中的存在是公认的，目前被认为是无创产前筛查和诊断的最佳靶点。然而，这些细胞的检测仍然存在问题，因为它们在产妇循环中的丰度较低，而且存在类似的母体来源的NRBC。目前鉴定胎儿NRBCs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)，随后的细胞形态分析，以及基于荧光原位杂交(FISH)或基于PCR的分子或遗传生物标记物的分析。然而，根据国家儿童健康和人类发育研究所的胎儿细胞分离研究(NIFTY)，这些方法都没有证明能够从母亲的血液中获得足够可靠的胎儿细胞，用于常规产前诊断。因此，开发一种从母血中分离胎儿NRBCs的有效方法是必要的，以便能够对其中少量的NRBCs进行常规检测，用于非侵入性产前诊断。我们推测，胎儿NRBCs的独特形态，以及随后其独特的介电性质，可以促进这些细胞的介电分离。这项建议描述了一种集成的设计和开发计划，该计划导致了一种新的小型化的介电激活细胞分选系统(DACS)，用于从母亲外周血中回收胎儿的NRBC。使用DACS系统分离胎儿NRBC将能够准确、早期地诊断和筛查染色体和遗传异常，而不会有流产的风险。此外，DACS系统可用于将其他类型的形态不同的细胞群与外周血分离，例如来自肿瘤或癌前病变的细胞群。公共卫生相关性：DACS技术的成功开发将带来一种高度敏感和特异的方法，用于非侵入性鉴定和分离胎儿NRBC，使其能够及时识别胎儿中的染色体和遗传异常，而不存在与标准程序相关的风险。然后，可以对一些已确定的产前疾病进行治疗，以改善未出生婴儿的前景。例如，生物素依赖和甲基丙二酸血症都是危及生命的遗传性疾病，已通过羊膜穿刺术诊断出来，并在子宫内进行了治疗，导致了健康婴儿的出生。当胎儿患有尚不可能进行产前治疗的情况时，产前诊断允许父母在情感上做好准备，并计划最安全的时机、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.