Improved fetal nucleated erythrocyte sorting purity using intracellular antifetal hemoglobin and Hoechst 33342

Fetal nucleated erythrocytes (FNRBC) flow sorted from maternal peripheral blood, using monoclonal antibodies (mAb) that bind fetal cell surface antigens, are a noninvasive source of fetal DNA for prenatal diagnosis. These mAbs, however, also bind antigens shared by maternal cells. In sorted populations, this results in maternal cell contamination and low fetal cell purities, which complicates genetic analysis by fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR). Fetal hemoglobin, (alpha 2 gamma 2), has been proposed as a useful fetal marker. To improve fetal cell enrichment from maternal blood, we developed an intracellular staining protocol that combines anti-gamma mAb with Hoechst 33342 to identify and flow sort FNRBC. Artificial mixtures of male umbilical cord cells (as a source of fetal hemoglobin) and female adult, non-pregnant peripheral blood mononuclear cells were stained and flow sorted using this protocol. FISH analysis was performed using chromosome X and Y specific probes. Fetal cell purities were calculated by microscope confirmation of anti-gamma staining and counting the number of X and Y signals present after FISH. Results from microscope analyses showed a fetal cell yield of 39-100% and fetal cell purities of 59-73%. These purities are significantly higher than the .001-4.8% previously reported by us in maternal samples using cell surface staining protocols. FISH results demonstrated that 83-100% (mean = 98%) of anti-gamma positive cells were male, whereas 82-100% (mean = 92.5%) of anti-gamma negative cells were female. These results confirmed that the anti-gamma mAb is highly fetal specific. When applied to maternal blood samples, this protocol should lead to increased sensitivity for prenatal diagnosis.