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 nucl eated red blood cells (NRBCs) isolated from the maternal circulation is ham pered by the low frequency of these cells in maternal blood, requiring exte nsive enrichment procedures before any analytical procedure can be performe d. 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 immunophenotypic ally identical to fetal NRBCs isolated from maternal blood. Magnetic activa ted 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 usi ng two-colour fluorescence in situ hybridization with X and Y chromosomal p robes. Using this model system, an NRBC isolation technique is described. I t 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 hi gher yield of male NRBCs than previously described MACS methodologies, in w hich CD71 positive cells were enriched after depletion of other cell types. Application of these isolation strategies to maternal blood samples result ed in a similar improved enrichment of male fetal cells after the direct en richment of CD71 positive cells. Copyright (C) 1999 John Wiley & Sons, Ltd.