28 May 2002 Combined FISH, anti-γ-Hb and DAPI for detection of fetal nucleated RBCs in maternal blood
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Proceedings Volume 4622, Optical Diagnostics of Living Cells V; (2002) https://doi.org/10.1117/12.468354
Event: International Symposium on Biomedical Optics, 2002, San Jose, CA, United States
Abstract
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 1:105 - 1:109 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-(gamma) -Hb fluorescence and DAPI staining was consistent and bright.
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Mona Farhad, Mona Farhad, Jeffrey H. Price, Jeffrey H. Price, } "Combined FISH, anti-γ-Hb and DAPI for detection of fetal nucleated RBCs in maternal blood", Proc. SPIE 4622, Optical Diagnostics of Living Cells V, (28 May 2002); doi: 10.1117/12.468354; https://doi.org/10.1117/12.468354
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