Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting.

Abstract

Fetal cells, potentially usable for prenatal diagnosis, were sorted from maternal blood samples taken as early as 15 weeks of gestation. Immunogenetic and cytogenic criteria established the fetal origin of the observed cells: Y-chromatincontaining (male) cells were detected in the sorted sample if and only if the newborn proved to be male and carried ceI1-surface antigens detected by the fluorescent-labeled antibody used for sorting with the fluorescence-activated cell sorter.Although the maternal-fetal barrier is generally considered to be effective in limiting cell transfer from fetus to mother, there have been several reports over the last 10 years suggesting that blood samples from pregnant women contain nucleated cells derived from the fetus. In these studies, putative male fetal cells were identified among maternal blood cells by the presence of either Y chromosome in metaphase spreads or Y chromatin after quinacrine staining of interphase nuclei. The presence of such male cells correlated reasonably well with births of male children, false negatives (births of male children but no Ychromatin-bearing cells) being the prevalent error. The conclusions from this work, however, received only qualified acceptance (reviewed in refs. 1 and 2), in part because the data relied solely on microscopic analyses requiring recognition of one Y-chromatin-positive cell per 1000-5000 maternal lymphocytes.In the studies presented here, we have added immunogenetic and cell-sorting techniques to the earlier methodology in order to improve the reliability of fetal cell identification in maternal blood samples. Using the fluorescence-activated cell sorter (FACS) (3, 4), we have sorted leukocytes from maternal blood and obtained samples enriched for cells that reacted with an antiserum specific for paternal antigens potentially carried by the fetus but not the mother. The enriched samples were then scored for cells containing Y chromatin, which could be derived only from a male fetus. The use of this enrichment technique facilitates detection of rare fetal cells in the maternal blood sample. More important, however, it provides an independent immunogenetic criterion for confirming the fetal origin of these cells-i.e., if Y-chromatin-containing cells were observed among those sorted from maternal blood, cells of the newborn from that pregnancy should carry the paternal antigens detected by the antiserum used for sorting. Thus, by examining FACS-enriched samples and correlating fetal cell detection with newborn reactivity with the sorting antiserum, we have definitely identified fetal cells in maternal circulation as early as the 15th week of gestation. MATERIALS AND METHODSFor the FACS-enrichment procedure, fetal cells among maternal peripheral blood lymphocytes were stained by indirect immunofluorescence by using first a rabbit antiserum directed against paternal HLA cell-surface antigens absent in the mother followed by a fluorescein-conjugated goat anti-rabbit immunoglobulin. HLA antibodies were chosen because (i) fetus...

Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84

“…In case of prenatal diagnosis such an approach would allow a rapid diagnosis of common trisomies even in cases where the preparation of metaphase chromosome spreads fails. Fetal cells in maternal blood (Herzenberg et al 1979) and tumor cytogenetics (Bloomfield 1985;LeBeau and Rowley 1986) provide obvious cases where the detection and quantitative evaluation of small subpopulations of cells bearing specific chromosomal aberrations should become feasible by interphase cytogenetics. Using fluorescence hybridization the detection of specific numerical chromosome aberrations can be combined with flow cytometry and fluorescence activated cell sorting (Trask et al 1985;Pinkel et al 1986).…”

Reproducing Genetics

“…Newly minted molecular technologies were evolving. Thus the concept of intact fetal cells in maternal blood for prenatal genetic diagnosis was reopened, following up the 1979 report of flow sorting of Y-chromatin cells from a pregnancy carrying a male fetus by Hertzenberg et al 10 In the late 1980s chromosome-specific fluorescent in situ hybridization became a practical option to interrogate fetal cells recovered from maternal blood. In collaboration with Kathy Klinger (Genzyme, Cambridge, MA), we used fluorescence-activated cell sorting to recover nucleated fetal red blood cells from maternal blood.…”

Flow cytometric methods for prenatal and neonatal diagnosis

“…Herzenberg first used fluorescence-activated cell sorting (FACS) to successfully isolate fetal cells in maternal blood in 1979 (Herzenberg et al, 1979). Using maternal peripheral blood samples, fetal cells were stained by indirect immunofluorescence using a rabbit antiserum targeting paternal HLA cell surface antigens absent in the mother followed by a fluorescein-conjugated goat anti-rabbit immunoglobulin.…”