Flow sorting of fetal erythroblasts using intracytoplasmic anti-fetal haemoglobin: preliminary observations on maternal samples.
Monoclonal antibody to fetal haemoglobin (alpha 2 gamma 2) has been proposed as a fetal-specific reagent. We developed an intracellular staining protocol that combines fluorescein isothiocyanate or phycoerythrin conjugated anti-gamma with the DNA binding dye Hoechst 33342 to identify and flow sort fetal erythroblasts from maternal blood. Our preliminary observations on anti-gamma-positive cells sorted from four different pregnant women are described here, using fluorescence in situ hybridization (FISH) with chromosome-specific probes to identify fetal cells. Our data demonstrate that far fewer candidate fetal cells are sorted with this protocol than by current cell surface staining methods that employ the monoclonal antibody CD71. This results in increased fetal cell sorting purities. With this protocol, standard FISH techniques require modification due to the rigorous fixation with 4 per cent paraformaldehyde. Our initial data indicate the promise of this approach.
Zheng
Y L
YL
Department of Pediatrics, New England Medical Center, Boston, MA 02111, USA.
Demaria
M
M
Zhen
D
D
Vadnais
T J
TJ
Bianchi
D W
DW
D000328:Adult; D000911:Antibodies, Monoclonal; D002469:Cell Separation / Q000379:methods*; D003593:Cytoplasm; D015342:DNA Probes; D004900:Erythroblasts / Q000166:cytology*; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005319:Fetal Hemoglobin / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008297:Male; D011247:Pregnancy; D014960:X Chromosome
D000911:Antibodies, Monoclonal; D015342:DNA Probes; D005456:Fluorescent Dyes; D005319:Fetal Hemoglobin; D016650:Fluorescein-5-isothiocyanate