SCANNING CYTOMETRY TO SORT FETAL NRBCS IN MATERNAL BLOOD

扫描细胞术对母血中的胎儿 NRBCS 进行分类

• 批准号: 6363436
• 负责人: JEFFREY H PRICE
• 金额: $ 13.15万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6363436
• 关键词: bioimaging /biomedical imaging; biomedical automation; biomedical equipment development; clinical research; erythrocytes; female; flow cytometry; human pregnant subject; image processing; imaging /visualization /scanning; pregnancy circulation

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.

在母体循环中发现罕见的胎儿成核红细胞（FNRBC）的发现导致了使用外围静脉穿刺的产前遗传筛查的想法。开发这种情况的动机一种最小的侵入性方法用于获得基因检测的胎儿细胞是降低成本和风险，并增加程序的可用性。遗传异常应通过超声，羊膜穿刺和绒毛膜绒毛采样来检测。尽管超声是非侵入性的，但许多遗传异常并未导致可检测的形态异常。因此，超声将与羊膜穿刺术或慢性绒毛采样（取决于胎龄），以筛查处于危险中的妊娠。这些技术涉及将针插入子宫，并带来胎儿的风险，其中包括很小的流产机会。简单的血液测试将代表一种低风险技术，其在广泛筛查中使用了巨大的潜力。利用周围孕产妇血液的挑战是估计的1/10,000,000 FNRBC。到目前为止，流式细胞术和其他分类方法未能达到这种稀有事件细胞选择所必需的准确性和吞吐量。此外，当前所有提出的方法都将富集步骤应用于含有Buffy Coat的NRBC。这些富集步骤进一步减少了FNRBC的数量。富集中FNRBC损失的可接受水平尚不清楚，因为无法直接测量母体外周血中数量（和变化）的直接测量。我们建议进一步开发基于图像的细胞分析技术。我们建议进一步开发基于图像的细胞分析技术（扫描细胞仪）至：1）直接测量居住在母体Buffy Coat中的胎儿FNRBC的数量，以及2）评估该技术作为产前基因筛查的常规细胞选择方法。扫描细胞术发展的两种策略 - 一种较慢，技术低风险的方法和一种未经测试的高速连续扫描方法，旨在进一步开发基于图像的原位分类。提出的大规模稀有事件检测的高性能扫描细胞仪具有极端广泛的临床和研究用途。修订（新字体）：主要的批评和修订是生物学的（仪器部分获得了出色的评论）。医学遗传学和鱼类专家Karen Arden博士被添加为研究者，以领导我们先前完善的DAPI/抗HBF-FITC双染色与双X和Y染色体鱼类染色的4色荧光技术。还将对FNRBCS与妊娠年龄的分布进行完整的统计分析，但是拟议的工作将首次直接直接测量FNRBC的数量，其中使用仪器具有具有很高的超级利率事件的精确度，该仪器在这项第一项研究的适当患者上具有很高的超级率事件精度。