PubMed · 8531786
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R C Brown, B E Lemmon. 1995. Methods in plant immunolight microscopy.. https://doi.org/10.1016/s0091-679x(08)61448-x
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To isolate fetal cells from maternal blood, we developed a new method based on galactose-bearing conjugation. Nucleated red blood cells (NRBCs), which highly express galactose on their surface, were selectively attached to a substrate coated with a galactose-containing polymer via soybean agglutinin (SBA), a galactose-specific lectin. Cord blood samples were used to evaluate enrichment efficacy of NRBCs by this method. Blood samples were obtained from 131 pregnant women between 6 and 27 gestational weeks. After preliminary condensation of fetal cells by Ficoll gradient centrifugation, NRBCs were enriched using galactose-positive selection by adjusting SBA concentration. We isolated one to several hundred NRBCs (mean+/-SD, 7.8+/-8.5) in 2.3 ml of peripheral blood samples from 96% of pregnant women. The isolated NRBCs were analyzed by a Y-chromosome FISH probe in eight cases carrying male fetuses. Y-signals were detected in all eight cases and more than half of the NRBCs were off fetal origin. The study demonstrates that our new method using galactose-specific lectin provides effective enrichment of fetal NRBCs allowing non-invasive prenatal diagnosis.
Fetal cells circulating in the peripheral blood of pregnant women are a potential target for noninvasive genetic analyses. They include epithelial (trophoblastic) cells, which are larger than peripheral blood leukocytes. We enriched circulating trophoblastic cells using the isolation by size of epithelial tumor cells (ISET) method. Peripheral blood was obtained at 11 to 12 weeks of pregnancy. Cells isolated by ISET were stained by hematoxylin and eosin or by immunohistochemistry. Large epithelial cells were microdissected and fetal cell identification was obtained by polymerase chain reaction with short tandem repeats and/or Y-specific primers. By analyzing only 2 ml of blood, we found a variable number (n = 1 to 7) of Y-positive cells (overall 15 of 23) in all of the six mothers carrying a male fetus. In contrast, none of the 26 cells isolated from seven mothers carrying a female fetus scored positive. Eleven cells were analyzed by using short tandem repeat-specific markers: six of them showed a fetal profile and five showed a maternal profile consistently with Y-specific results. Only one-fifth of the single cell DNA was used for fetal cell assessment, leaving enough material for further genetic tests. We also show that the ISET approach allows the performance of fluorescence in situ hybridization analyses and the detection of DNA point mutations in single microdissected cells. We conclude that this is a powerful approach to enrich circulating fetal cells and prove their fetal origin, and that it may have implications for noninvasive prenatal diagnosis of genetic disorders.
T-helper (Th) 2-type cytokines predominate in decidua, plausibly accounting for protection of a semiallograft, the embryo and placenta, from attack by the maternal immune system. However, localization of Th2 and T-cytotoxic (Tc) 2 cells in decidua has not been reported, presumably because of the difficulty in detecting intracellular cytokines in tissues. Here, by staining tissues for a novel surface maker of Th2/Tc2, the chemoattractant receptor-homologous molecule CRTH2, which is expressed on Th2 cells, we show that CRTH2(+) Th2 cells and CRTH2(+) Tc2 cells are significantly increased at the materno-fetal interface (implantation site) in decidua. We also show that trophoblast, uterine epithelium and endometrial glands all express haematopoietic-type prostaglandin (PG) D(2) synthase (hPGDS). Since CRTH2 is a chemoattractant receptor for PGD(2) and mediates PGD(2)-dependent migration of blood Th2 cells, our findings suggest that Th2 and Tc2 cells may be recruited to the materno-fetal interface, at least in part in a PGD(2)-mediated manner.