Isolation of fetal DNA from nucleated erythrocytes in maternal blood.

Abstract

Fetal nucleated cells within maternal blood represent a potential source of fetal genes obtainable by venipuncture. We used monoclonal antibody against the transferrin receptor (TfR) to identify nucleated erythrocytes in the peripheral blood of pregnant women. Candidate fetal cells from 19 pregnancies were isolated by flow sorting at 121/-17 weeks gestation. The DNA in these cells was amplified for a 222-base-pair (bp) sequence present on the short arm of the Y chromosome as proof that the cells were derived from the fetus. The amplified DNA was compared with standardized DNA concentrations; 0.1-1 ng offetal DNA was obtained in the 20-ml maternal samples. In 7/19 cases, a 222-bp band of amplified DNA was detected, consistent with the presence of male DNA in the isolated cells; 6/7 of these were confirmed as male pregnancies by karyotyping amniocytes. In the case of the female fetus, DNA prepared from samples at 32 weeks of gestation and cord blood at delivery also showed the presence of the Y chromosomal sequence, suggesting Y sequence mosaicism or translocation. In 10/12 cases where the 222-bp band was absent, the fetuses were female. Thus, we were successful in detecting the Y chromosomal sequence in 75% of the male-bearing pregnancies, demonstrating that it is possible to isolate fetal gene sequences from cells in maternal blood. Further rermement in methodology should increase sensitivity and facilitate noninvasive screening for fetal gene mutations.

Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma

“…For the direct detection of fetal chromosomal and genetic abnormalities from maternal blood, early work focused on the relatively difficult isolation of the rare fetal nucleated cells from maternal blood (3)(4)(5). The discovery of cell-free fetal nucleic acids in maternal plasma in 1997 opened up new possibilities (6,7).…”

Isolating highly enriched populations of circulating epithelial cells and other rare cells from blood using a magnetic sweeper device

“…Applications range from characterization of circulating epithelial cells (CEpCs) in the peripheral blood of cancer patients for disease prognosis and personalized treatment (1)(2)(3)(4)(5)(6)(7); circulating fetal cells in maternal blood for prenatal diagnosis (8)(9)(10); and antigen-specific lymphocytes for immune monitoring (11). The deconvolution of profiling data to extract the relevant biology of rare cells from the mixture of blood leukocytes is challenging, and in most cases impractical (12,13).…”

A comparison of the choice of monoclonal antibodies for recovery of fetal cells from maternal blood using FACS for noninvasive prenatal diagnosis of hemoglobinopathies

“…Bianchi et al, 1990 using CD71 were able to detect the Y chromosomal sequence in 75% of the male-bearing pregnancies in 19 women (1). In 1995, Bianchi et al also showed that CD71 selection using FACS gave the best and consistent results as compared to immunomagnetic beads and MACS (11).…”

“…Many methods like Fluorescence Activated Cell Sorting (FACS), Magnetic Activated Cell Sorting (MACS), charge flow separation, immunofluorescent beads, lectins, etc. have been exploited for the isolation of fetal nucleated red blood cells (NRBCs) from maternal peripheral blood (1)(2)(3)(4)(5). Of these, FACS has been the most widely used technique.…”