SCANNING CYTOMETRY TO SORT FETAL NRBCS IN MATERNAL BLOOD

在母体循环中发现罕见的胎儿有核红细胞（fnRBCS）导致了使用外周静脉穿刺进行产前遗传筛查的想法。开发这种获取胎儿细胞用于基因检测的微创方法的动机是降低成本和风险，并增加程序的可用性。遗传异常将通过超声、羊膜穿刺术和绒毛膜绒毛取样来检测。虽然超声是非侵入性的，但许多遗传异常不会导致可检测的形态异常。因此，超声结合羊膜穿刺术或慢性绒毛取样（取决于胎龄）筛查高危妊娠。这些技术需要将一根针插入子宫，对胎儿有风险，包括流产的可能性很小。简单的血液检查是一种低风险的技术，具有广泛筛查的巨大潜力。利用外周血的挑战在于定位大约1/ 1000000的fnrbc。到目前为止，流式细胞术和其他分选方法都未能达到这种罕见事件细胞选择所需的准确性和吞吐量。此外，目前提出的所有方法都对含有nRBC的灰褐色被膜进行富集处理。这些富集步骤进一步减少了fnrbc的数量。由于不可能直接测量母体外周血中fnRBC的数量（和变化），因此在富集过程中fnRBC损失的可接受水平尚不清楚。我们建议进一步发展基于图像的细胞分析技术。我们建议进一步发展基于图像的细胞分析技术（扫描细胞术），以：1)直接测量驻留在母体灰白色被中的胎儿fnrbc的数量；2)评估该技术作为产前遗传筛查的常规细胞选择方法。为进一步发展基于图像的原位分选，提出了扫描细胞术发展的两种策略——一种速度较慢、技术风险较低的方法和一种未经测试的高速连续扫描方法。所提出的用于大规模罕见事件检测的高性能扫描细胞术具有极其广泛的临床和研究应用潜力。修订（新字体）：主要的批评和修订是生物学的（仪器部分得到了很好的评价）。医学遗传学和FISH专家Karen Arden博士作为研究员加入，领导将我们先前完善的DAPI/anti- HbF-FITC双染色与双X和Y染色体FISH染色相结合，用于四色荧光技术。还将对fnrbc与妊娠年龄的分布进行全面的统计分析，但一项拟议的工作将提供fnrbc数量的第一次直接测量，使用一种仪器在适当数量的患者中证明具有高超高率事件准确性。

The finding of rare fetal nucleated red blood cells (fnRBCS) in the maternal circulation has led to the idea of developing prenatal genetic screening using peripheral venipuncture. The motivations for developing this a minimally invasive method for obtaining fetal cells for genetic testing are to reduce cost and risk, and increase the availability of the procedure. Genetic anomalies are to be detected by ultrasound, amniocentesis and chorionic villus sampling. Although ultrasound is non- invasive, many genetic abnormalities do not result in detectable morphological abnormalities. Thus, ultrasound is combined with amniocentesis or chronic villus sampling (depending on gestational age) for screening at-risk pregnancies. These techniques involve inserting a needle into the uterus and entail risks to the fetus that include a small chance of miscarriage. A simple blood test would represent a low-risk technique with tremendous potential for use in widespread screening. The challenge in utilizing peripheral maternal blood is locating the estimated 1/10,000,000 fnRBCs. Thus far, flow cytometry and other sorting methods have failed to achieve the accuracy and throughput necessary for this magnitude of rare event cell selection. In addition, all currently proposed methods apply enrichment steps to the nRBC- containing buffy coat. These enrichment steps further reduce the number of fnRBCs. The acceptable level of fnRBC loss in enrichment is unknown because the direct measurements of the numbers (and variations) in maternal peripheral blood have not been possible. We propose to further develop image-based cell analysis technology. We propose to further develop image-based cell analysis technology (scanning cytometry) to: 1) measure directly the number of fetal fnRBCs residing in the maternal buffy coat, and 2) evaluate the technology as a routine cell selection method for prenatal genetic screening. Two strategies for scanning cytometry development- a slower, technologically low-risk method and a more untested high-speed continuous-scanning method-are proposed for further development of image-based in situ sorting. The proposed high-performance scanning cytometry for large-scale rare event detection has potential for extremely broad clinical and research use. Revisions (in new font): The primary critiques and revisions are biological (the instrumentation portion received excellent reviews). Dr. Karen Arden, an medical genetics and FISH expert, was added as an investigator to lead the combining of our previously perfected DAPI/anti- HbF-FITC dual stain with a dual X and Y chromosome FISH stain for a 4-color fluorescence technique. Full statistical analysis of the distribution of fnRBCs vs. gestation age will also be performed, but a proposed work will, however, provide the first direct measurement of the numbers of fnRBCs using an instrument with demonstrated high ultra-rate event accuracy on an appropriate number of patients for this first study.