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首页> 外文会议>Conference on Optical Diagnostics of Living Cells Ⅴ, Jan 23-25, 2002, San Jose, USA >Combined FISH, Anti-γ-Hb and DAPI for Detection of Fetal Nucleated RBCs in Maternal Blood
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Combined FISH, Anti-γ-Hb and DAPI for Detection of Fetal Nucleated RBCs in Maternal Blood

机译：结合FISH，抗γ-Hb和DAPI检测母体血液中的胎儿有核红细胞

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摘要

Since the 1970s, extensive research has been devoted to the development of a standard procedure for the isolation of fetal nucleated red cells (fnRBCs) from maternal blood. Since these cells are sources of fetal DNA, cytogenetic analysis would lead to a minimally-invasive method for the prenatal diagnosis of chromosomal and genetic disorders early in gestation. FnRBCs constitute a significant portion of the fetal blood, have a short and finite life span, and are rare in peripheral adult blood. They have been reported to exist in the maternal circulation at frequencies as low as 1:10~5-1:10~9 maternal nucleated cells. Due to these ultra-rare frequencies, isolation with minimal loss has been a time and labor-intensive process. To overcome this problem, a fully automated scanning cytometer that incorporates high-performance autofocus and image segmentation has been built and shown higher rate, quantity, sensitivity (true positive rate) and specificity (true negative rate) in a model cell preparation. For detecting fnRBCs, two discriminating characteristics may suffice: 1) the presence of fetal hemoglobin, which is the major intracytoplasmic protein found in fetal red cells from 5 to 35 weeks gestation, and 2) the presence of a nucleus. In clinical trials, the fetal origin of the isolated cells will be confirmed by fluorescence in situ hybridization (FISH) on the X and Y chromosomes in male pregnancies. The aim of the present study was to develop a reliable and reproducible staining method for combined immunofluorescence and FISH analysis for these clinical trials. This staining technique was developed using fnRBCs extracted from fetal liver blood and a human erythroleukemia cell line (HEL) that expresses fetal hemoglobin. The resulting method for four-color X- and Y-FISH, anti-γ-Hb fluorescence and DAPI staining was consistent and bright.
机译：自1970年代以来，广泛的研究致力于开发从母体血中分离胎儿有核红细胞（fnRBC）的标准程序。由于这些细胞是胎儿DNA的来源，细胞遗传学分析将导致一种微创方法，可在妊娠早期对染色体和遗传疾病进行产前诊断。 FnRBC占胎儿血液的很大一部分，寿命短而有限，在成年外周血中很少见。据报道它们存在于母体循环中，其频率低至1：10〜5-1：10〜9母体有核细胞。由于这些极少的频率，以最小的损耗进行隔离已经成为时间和劳动密集的过程。为了克服这个问题，已经建立了一种结合了高性能自动聚焦和图像分割功能的全自动扫描细胞仪，并在模型细胞制备中显示出更高的速率，数量，灵敏度（真阳性率）和特异性（真阴性率）。为了检测fnRBC，两个鉴别特征就足够了：1）胎儿血红蛋白的存在，它是在妊娠5至35周的胎儿红细胞中发现的主要胞浆内蛋白，以及2）核的存在。在临床试验中，分离的细胞的胎儿起源将通过男性怀孕的X和Y染色体上的荧光原位杂交（FISH）来证实。本研究的目的是为这些临床试验开发一种结合免疫荧光和FISH分析的可靠且可重复的染色方法。使用从胎儿肝血中提取的fnRBC和表达胎儿血红蛋白的人类红血球细胞系（HEL）开发了这种染色技术。所得的四色X-和Y-FISH，抗γ-Hb荧光和DAPI染色的方法一致且明亮。

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