The use of in vitro expanded erythroid cells in a model system for the isolation of fetal cells from maternal blood.
The development of a non-invasive prenatal diagnostic test using fetal nucleated red blood cells (NRBCs) isolated from the maternal circulation is hampered by the low frequency of these cells in maternal blood, requiring extensive enrichment procedures before any analytical procedure can be performed. In order to improve and simplify these procedures, we have used in vitro expanded erythroid cells derived from male umbilical cord blood in a model system for the isolation of fetal NRBCs from maternal blood. Erythroblast cells were expanded in vitro to high cell numbers and were immunophenotypically identical to fetal NRBCs isolated from maternal blood. Magnetic activated cell sorting (MACS) isolation procedures were optimized using in vitro expanded male NRBCs diluted up to 1 in 400,000 with female peripheral blood mononucleated cells. The number of recovered male cells was determined using two-colour fluorescence in situ hybridization with X and Y chromosomal probes. Using this model system, an NRBC isolation technique is described. It is based on a one-step MACS enrichment protocol for CD71 positive cells, which showed a significant (Wilcoxon signed ranks test, p<0.05) two-fold higher yield of male NRBCs than previously described MACS methodologies, in which CD71 positive cells were enriched after depletion of other cell types. Application of these isolation strategies to maternal blood samples resulted in a similar improved enrichment of male fetal cells after the direct enrichment of CD71 positive cells.
D015703:Antigens, CD / Q000032:analysis; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D004900:Erythroblasts / Q000166:cytology* / Q000276:immunology; D004921:Erythropoietin / Q000494:pharmacology; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D005865:Gestational Age; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007231:Infant, Newborn; D017493:Leukocyte Common Antigens / Q000032:analysis; D008280:Magnetics; D008297:Male; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods; D011990:Receptors, Transferrin; D019089:Stem Cell Factor / Q000494:pharmacology; D014960:X Chromosome; D014998:Y Chromosome