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...

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The incorporation of iron into human cells involves the binding of diferric transferrin to a specific cell surface receptor. We studied the process of endocytosis in K562, a human erythroid cell line, by using tetramethylrhodamine isothiocyanate-labeled transferrin (TRITC-transferrin) and fluorescein isothiocyanate-labeled Fab fragments of goat antireceptor IgG preparation (FITC-Fabantitransferrin receptor antibody). Because the antireceptor antibody and transferrin bind to different sites on the transferrin receptor molecule it was possible to simultaneously and independently follow ligand and receptor. At 4°C, the binding of TRITC-transferrin or FITC-Fab antitransferrin receptor antibody exhibited diffuse membrane fluorescence. At 20°C, the binding of TRITC-transferrin was followed by the rapid formation of aggregates. However, the FITC-Fab antitransferrin receptor did not show similar aggregation at 20°C unless transferrin was present. In the presence of transferrin, the FITC-Fab antitransferrin receptor antibody formed aggregates at the same sites and within the same time period as TRITC transferrin, indicating co-migration. Although the diffuse surface staining of either label was removed by proteolysis, the larger aggregates were not susceptible to enzyme degradation, indicating that they were intracellular. The internal location of the aggregates was also demonstrated using permeabilized cells that had been preincubated with transferrin and fixed with 4% paraformaldehyde. These cells showed aggregated receptor in the interior of the cell when reacted with fluorescein-labeled antibody to the receptor. This indicated that the transferrin and the transferrin receptor co-internalize and migrate to the same structures within the cell.Transferrin, an iron-binding serum protein, and its receptor on the plasma membrane are considered to be involved in a major pathway for the transport of iron into cells. Superficially, the receptor appears to act as a cation transporter; however, the detailed mechanism of the cellular internalization of iron is still unclear. Initial studies using ~3q-labeled transferrin and cell fractionation found transferfin on the surface of reticuiocytes (l). In more recent work, using ascites tumor cells, ferrocyanide staining of transferrin and ferritinconjugated antibody to transferrin also showed that transferfin is localized on the cell surface (2). In contrast, there is evidence that transferrin is pinocytosed into reticulocytes and normoblasts (3)(4)(5). In these studies, ferritin-conjugated antibodies to transferrin (3, 4) and transferrin-colloidal gold complexes (5) were employed to visualize the localization of transferrin.Experiments demonstrating that transferrin co-purifies with clathrin in h u m a n placental tissues (6, 7) imply that the mechanism of transferrin uptake into cells might be similar to that of many peptides, i.e., asialoglycoprotein receptor, yolk proteins, a-2 macroglobulin, and the hormones, insulin and epidermal growth factor (8, 9). Many studies of r...

The fluorescence of the human Y chromosome after staining with quinacrine derivatives was used in an attempt to determine the prevalence of XY lymphocytes presumptively emanating from male fetuses in the blood of pregnant women. Seven women who subsequently gave birth to male infants had 0.03%-0.20% lymphocytes with a Y body. In two women pregnant with male fetuses, no Y bodies were detected. Of 12 women who gave birth to girls, eight had no Y bodies. In the remaining four, fluorescent spots were interpreted as Y bodies. One of those women had previously had an abortion. In the remaining three cases, it was found in retrospect that the fluorescent spots emanated from exceptionally brightly fluorescent autosomal regions in the maternal or paternal chromosomes, or both. Phytohemagglutinin-stimulated and unstimulated lymphocytes showed similar frequencies of Y body. Based on determinations of the incidence of Y body-containing lymphocytes in males (35%-66.5%), it was estimated that approximately 0.1%-0.5% of lymphocytes in the blood of most women pregnant with a male fetus are of fetal origin. This figure is remarkably high, since it exceeds in relative terms that of fetal erythrocytes (which mostly appear in the mother’s blood at the time of delivery) by several orders of magnitude. It is suggested that fetal lymphocytes actively cross the feto-maternal barriers. This phenomenon may have a profound influence on the immunologic interactions between fetus and mother. The method described may not turn out to be profitable in the assessment of the fetal sex, since its accuracy may be of the order of only 80%-95%. Moreover, several limitations prevail.