Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization

Abstract

Abstract 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 γ-globin are problematic. They are nonspecific because maternal cells may produce γ-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 ε-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 ε-positive fetal erythroblast was identified among 105 maternal nucleated cells in 6 paired mixture experiments of fetal erythroblasts in maternal blood (P < .001). Male ε-positive fetal erythroblasts were clearly distinguishable from adult female ε-negative erythroblasts, with no false positives (n = 1000). The frequency of fetal erythroblasts expressing ε-globin declines linearly from 7 to 14 weeks' gestation (y = −15.8 × + 230.8;R2 = 0.8; P < .001). We describe a rapid and accurate method to detect simultaneously fetal erythroblast morphology, intracytoplasmic ε-globin, and nuclear FISH.