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

Isolating fetal erythroblasts from maternal blood offers a promising noninv asive alternative for prenatal diagnosis. The current immunoenzymatic metho ds of identifying fetal cells from background maternal cells postenrichment by labeling gamma -globin are problematic. They are nonspecific because ma ternal cells may produce gamma -globin, give poor hybridization efficiencie s with chromosomal fluorescence in situ hybridization (FISH), and do not pe rmit simultaneous visualization of the fetal cell identifier and the FISH s ignal. We describe a novel technique that allows simultaneous visualization of fetal erythroblast morphology, chromosomal FISH, and is an element of - globin labeled with AMCA (7-amino-4-methylcoumarin-3-acetic acid). AMCA was chosen as the fluorescent label to circumvent the problem of heme autofluo rescence because the mean difference in relative fluorescence intensity bet ween fetal erythroblasts stained positive for antiglobin antibody and autof luorescence of unstained cells was greater with AMCA (mean 43.2; 95% confid ence interval [CI], 34.6-51.9; SD = 14.0) as the reporting label compared w ith fluorescein isothiocyanate (mean 24.2; 95% Cl, 16.4-31.9; SID = 12.4) o r phycoerythrin (mean 9.8; 95% Cl, 4.8-14.8; SID = 8.0). Median FISH hybrid ization efficiency was 97%, comparable to the 98% (n = 5 paired samples) us ing Carnoy fixative. One is an element of -positive fetal erythroblast was identified among 10(5) maternal nucleated cells in 6 paired mixture experim ents of fetal erythroblasts in maternal blood (P < .001). Male E-positive f etal erythroblasts were clearly distinguishable from adult female E-negativ e erythroblasts, with no false positives (n = 1000). The frequency of fetal erythroblasts expressing is an element of -globin declines linearly from 7 to 14 weeks' gestation (y = -15.8 x +230.8; R-2 = 0.8; P < .001). We descr ibe a rapid and accurate method to detect simultaneously fetal erythroblast morphology, intracytoplasmic e-globin, and nuclear FISH.