Detection of Apoptotic Fetal Cells in Plasma of Pregnant Women

Abstract

Fetal DNA is present in plasma of pregnant women in amounts almost 1000-fold higher than the DNA present in intact fetal cells circulating in maternal peripheral blood (1)(2). The clinical usefulness of fetal DNA from plasma for noninvasive prenatal diagnosis has been demonstrated recently through analysis of the fetal RhD status by PCR-based approaches (3)(4)(5). We explored the possibility that not all of this fetal DNA is soluble and cell-free, but that part of it is still cell-associated. Heparin blood samples of 25–30 mL were obtained after informed consent from 38 pregnant women attending the Prenatal Diagnostic Center at week 7–16 of gestation. Several weeks after blood withdrawal, chorionic villus sampling or amniocentesis was performed as planned at each patient’s first visit. The protocol was approved by the Committee of Medical Ethics of the University Hospital ‘Vrije Universiteit’. Blood samples were processed on the day of withdrawal and were centrifuged on a discontinuous Percoll gradient as described previously (6)(7). In brief, after dilution of the heparin blood in Hanks’ balanced salt solution; 1:2 dilution (Life Technologies), blood was layered on a 5-step Percoll density gradient consisting of 15 mL of 600 mL/L and 5 mL each of 550, 500, 450, and 400 mL/L Percoll in Hanks’ balanced salt solution. After centrifugation for 25 min at 1000 g at room temperature, plasma samples were removed from the upper part …