NONINVASIVE PRENATAL-DIAGNOSIS - USE OF DENSITY GRADIENT CENTRIFUGATION, MAGNETICALLY ACTIVATED CELL SORTING AND IN-SITU HYBRIDIZATION

OBJECTIVE: To develop a noninvasive method suitable for clinical prena tal diagnosis. STUDY DESIGN: Fetal nucleated erythrocytes were separat ed from peripheral blood of 17 healthy pregnant women using small magn etically activated cell sorting columns (MiniMACS) following density g radient centrifugation and dual antibody labeling methods. The protoco l was designed to compare the efficacy of antitransferrin receptor (CD 71)/antiglycophorin A (GPA) antibodies with antithrombospondin recepto r (CD36)/anti-GPA antibodies in identifying nucleated erythrocytes in maternal blood. Cytospin preparations of the isolated cells were subje cted to in situ hybridization with specific DNA probes for the Y chrom osome and chromosome 21 to confirm the fetal origin. RESULTS: After Mi niMACS the enrichment factors for the CD71/GPA- and CD36/GPA-positive cells from maternal blood were similar, and the percentages of fetal c ells recovered did not differ. Seven of seven male pregnancies were co rrectly identified. One case of trisomy 21 was detected. CONCLUSION: T he in situ hybridization analysis of fetal nucleated erythrocytes isol ated from maternal blood using single density gradient centrifugation, anti-CD71/anti-GPA immunostaining and MiniMACS could be an accurate, sensitive and noninvasive method for prenatal diagnosis.