Choolani M, O'Donnell H, Campagnoli C, Kumar S, Roberts I, Bennett P R, Fisk N M
Department of Maternal and Fetal Medicine, Division of Paediatrics, Institute of Reproductive and Developmental Biology, Imperial College School of Medicine, Hammersmith Hospital Campus, Du Cane Rd., London W12 0NN, United Kingdom.
Blood. 2001 Aug 1;98(3):554-7. doi: 10.1182/blood.v98.3.554.
Isolating fetal erythroblasts from maternal blood offers a promising noninvasive alternative for prenatal diagnosis. The current immunoenzymatic methods of identifying fetal cells from background maternal cells postenrichment by labeling gamma-globin are problematic. They are nonspecific because maternal cells may produce gamma-globin, give poor hybridization efficiencies with chromosomal fluorescence in situ hybridization (FISH), and do not permit simultaneous visualization of the fetal cell identifier and the FISH signal. We describe a novel technique that allows simultaneous visualization of fetal erythroblast morphology, chromosomal FISH, and epsilon-globin labeled with AMCA (7-amino-4-methylcoumarin-3-acetic acid). AMCA was chosen as the fluorescent label to circumvent the problem of heme autofluorescence because the mean difference in relative fluorescence intensity between fetal erythroblasts stained positive for antiglobin antibody and autofluorescence of unstained cells was greater with AMCA (mean 43.2; 95% confidence interval [CI], 34.6-51.9; SD = 14.0) as the reporting label compared with fluorescein isothiocyanate (mean 24.2; 95% CI, 16.4-31.9; SD = 12.4) or phycoerythrin (mean 9.8; 95% CI, 4.8-14.8; SD = 8.0). Median FISH hybridization efficiency was 97%, comparable to the 98% (n = 5 paired samples) using Carnoy fixative. One epsilon-positive fetal erythroblast was identified among 10(5) maternal nucleated cells in 6 paired mixture experiments of fetal erythroblasts in maternal blood (P <.001). Male epsilon-positive fetal erythroblasts were clearly distinguishable from adult female epsilon-negative erythroblasts, with no false positives (n = 1000). The frequency of fetal erythroblasts expressing epsilon-globin declines linearly from 7 to 14 weeks' gestation (y = -15.8 x + 230.8; R(2) = 0.8; P <.001). We describe a rapid and accurate method to detect simultaneously fetal erythroblast morphology, intracytoplasmic epsilon-globin, and nuclear FISH. (Blood. 2001;98:554-557)
从母体血液中分离胎儿成红细胞为产前诊断提供了一种有前景的非侵入性替代方法。目前通过标记γ-珠蛋白从富集后的母体背景细胞中鉴定胎儿细胞的免疫酶法存在问题。它们是非特异性的，因为母体细胞可能产生γ-珠蛋白，与染色体荧光原位杂交（FISH）的杂交效率低，并且不允许同时可视化胎儿细胞标识符和FISH信号。我们描述了一种新技术，该技术允许同时可视化胎儿成红细胞形态、染色体FISH以及用AMCA（7-氨基-4-甲基香豆素-3-乙酸）标记的ε-珠蛋白。选择AMCA作为荧光标记以规避血红素自发荧光的问题，因为与异硫氰酸荧光素（均值24.2；95%置信区间[CI]，16.4 - 31.9；标准差 = 12.4）或藻红蛋白（均值9.8；95%CI，4.8 - 14.8；标准差 = 8.0）相比，以AMCA作为报告标记时，抗球蛋白抗体染色阳性的胎儿成红细胞与未染色细胞的自发荧光之间相对荧光强度的平均差异更大（均值43.2；95%CI，34.6 - 51.9；标准差 = 14.0）。FISH杂交效率中位数为97%，与使用卡诺固定剂时的98%（n = 5对样本）相当。在6对母体血液中胎儿成红细胞的混合实验中，在10⁵个母体有核细胞中鉴定出1个ε-阳性胎儿成红细胞（P <.001）。雄性ε-阳性胎儿成红细胞与成年雌性ε-阴性成红细胞明显可区分，无假阳性（n = 1000）。表达ε-珠蛋白的胎儿成红细胞频率在妊娠7至14周呈线性下降（y = -15.8x + 230.8；R² = 0.8；P <.001）。我们描述了一种快速准确的方法，可同时检测胎儿成红细胞形态、胞质内ε-珠蛋白和核FISH。（《血液》。2001年；98：554 - 557）
