Cultivation of fetal erythroid precursors from maternal blood: Isolation and characterization by PCR and FISH

Abstract

Cultivation of fetal progenitor cells from maternal blood offers the opportunity to produce sufficient fetal cells for prenatal molecular genetic and cytogenetic analysis. For in vitro cultivation, 10 ml of blood was collected from 22 women carrying a male fetus. After triple-density gradient centrifugation, the mononucleated cells were cultivated for 10 to 14 days in special hematopoietic growth medium. Red and white colored cell colonies were individually collected by micromanipulation. A representative sample from each colony was characterized by chromosome Y-specific polymerase chain reaction (PCR) systems. The remaining cells of the Y-positive colonies were used to perform chromosome preparations and fluorescence in situ hybridization (FISH) to detect XY-positive interphase nuclei and metaphases. Y-positive signals could be detected in 15 (68%) of the 22 analyzed blood samples. With SRY PCR 10.5% (40/379) of the collected red colonies were determined to be of fetal origin and 6.1% (32/522) of the colonies analyzed by amelogenin PCR were Y-positive. All collected white cell colonies were Y-negative. FISH analyses of PCR-positive colonies revealed that less than 30% of the cells within a colony are of fetal origin and reflect more precisely the actual situation within a single colony. Moreover the successful preparation of fetal metaphases in non-invasive prenatal diagnosis is presented.