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Blood, 1 August 2001, Vol. 98, No. 3, pp. 554-557
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Simultaneous fetal cell identification and diagnosis by
epsilon-globin chain immunophenotyping and chromosomal fluorescence in
situ hybridization
Mahesh Choolani,
Hilary O'Donnell,
Cesare Campagnoli,
Sailesh Kumar,
Irene Roberts,
Phillip R. Bennett, and
Nicholas M. Fisk
From the Department of Maternal and Fetal Medicine,
Division of Paediatrics, Obstetrics and Gynaecology, Institute of
Reproductive and Developmental Biology, and Department of Haematology,
Imperial College School of Medicine, Hammersmith Hospital Campus,
London, United Kingdom.
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  -globin are problematic. They are nonspecific because maternal cells may produce -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  -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 -positive fetal erythroblast was identified among 105 maternal nucleated cells in 6 paired mixture
experiments of fetal erythroblasts in maternal blood
(P < .001). Male -positive fetal erythroblasts were
clearly distinguishable from adult female -negative erythroblasts,
with no false positives (n = 1000). The frequency of fetal
erythroblasts expressing -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 -globin, and nuclear FISH.
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