Prenatal genetic diagnosis from maternal blood: Simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative and positive magnetic activated cell sorting

Fecal nucleated red blood cells (nRBCs) are rare in maternal circulation, b ut their presence constitutes a potential source of non-invasive prenatal g enetic diagnosis. This study was undertaken to establish a non-invasive pre natal genetic diagnosis method using isolated fetal nRBCs. A multi-step met hod including triple density gradient and magnetic activated cell sorting ( MACS) using CD45 and CD71, cytospin centrifugation, K-B staining, and glyco phorin A-immuno fluorescence in situ hybridization (GPA-immuno FISH) was pe rformed. The study population included 65 patients From 8 to 41 weeks of ge station, and fetal nRBC was separated from all cases. The number of fetal n RBCs retrieved was 12.8+/-2.7 in 8 to 11 gestational weeks, 15.2+/-6.5 in 1 2 to 18 gestational weeks, 16.4+/-6.5 in 19 to 23 gestational weeks, 10.6+/ -3.2 in 24 to 28 gestational weeks, and 5.5+/-1.9 in 35 to 41 gestational w eeks: the mean number of nRBCs collected from 20 mi of maternal peripheral blood was 13.7+/-6.2. The highest value of yield was 45.6% from 12 to 18 we eks gestation. The fetal sex determination confirmed by amniocentesis or ch orionic villus sampling showed 100% sensitivity and 91.7% specificity for m ates; 91.7% sensitivity and 100% specificity for females. We showed that fe tal cells can be reliably enriched from maternal blood and that they can be used for detecting specific chromosomes by FISH with a specificity superio r to current non-invasive methods.