Lewis D E, Schober W, Murrell S, Nguyen D, Scott J, Boinoff J, Simpson J L, Bischoff F Z, Elias S
Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX, USA.
Cytometry. 1996 Mar 1;23(3):218-27. doi: 10.1002/(SICI)1097-0320(19960301)23:3<218::AID-CYTO5>3.0.CO;2-E.
A noninvasive method of prenatal genetic diagnosis requires fetal cell selection from the maternal circulation that allows efficient recovery for analysis by fluorescence in situ hybridization (FISH). We have solved several problems that negatively affect the isolation and FISH analysis of fetal nucleated red blood cells (nRBCs) in the maternal circulation. The use of glycophorin A (Gly A) antibodies (Abs) for selection is problematic because all five monoclonal antibodies (mAbs) tested caused agglutination of non-nRBCs, thereby changing both light scatter and fluorescence properties of cells by flow cytometry. Because the number of non-nRBCs is variable after Ficoll separation, isolation of nRBCs could be compromised severely by agglutination of nucleated cells with nonnucleated cells, causing them to shift light scatter and fluorescence properties. Several methods for the removal of unwanted maternal white blood cells with CD45 mAbs were also evaluated. Magnetic bead depletion was found to interfere with FISH detection because of residual bead debris after sorting. By contrast, removal of CD45+ cells by a panning technique eliminated this problem. Positive selection methods based on CD71, CD45, and LDS-751 staining and detection of fetal cells by gamma globin expression were also analyzed. Fetal cells were detected by FISH in 11 of 19 (CD71 selection) and in 13 of 15 (gamma selection) random pregnancies. These data support the possibility of a noninvasive method for isolation and analysis of fetal cells for prenatal diagnosis.
一种非侵入性产前基因诊断方法需要从母体循环中筛选胎儿细胞，以便能有效地回收细胞，用于荧光原位杂交（FISH）分析。我们已经解决了几个对母体外周血中胎儿有核红细胞（nRBCs）的分离及FISH分析产生负面影响的问题。使用血型糖蛋白A（Gly A）抗体（Abs）进行筛选存在问题，因为所测试的全部5种单克隆抗体（mAbs）都会引起非nRBCs的凝集，从而通过流式细胞术改变细胞的光散射和荧光特性。由于Ficoll分离后非nRBCs的数量可变，有核细胞与无核细胞的凝集可能会严重影响nRBCs的分离，导致它们的光散射和荧光特性发生改变。我们还评估了几种使用CD45 mAbs去除不需要的母体白细胞的方法。发现磁珠去除法会干扰FISH检测，因为分选后会残留磁珠碎片。相比之下，通过淘选技术去除CD45+细胞则消除了这个问题。我们还分析了基于CD71、CD45和LDS-751染色的阳性选择方法以及通过γ珠蛋白表达检测胎儿细胞的方法。在19例随机妊娠中有11例（CD71选择法）以及15例中有13例（γ选择法）通过FISH检测到了胎儿细胞。这些数据支持了采用非侵入性方法分离和分析胎儿细胞用于产前诊断的可能性。
