Fetal erythroblasts from maternal blood identified with 2,3-bisphosphoglycerate (BPG) and in situ hybridization (ISH) using Y-specific probes

Different types of fetal nucleated cells can be found in maternal blood, providing the possibility of non-invasive prenatal diagnosis. For this purpose, we have studied fetal erythroblasts. We discovered that haemoglobin-containing cells treated with 2,3-bisphosphoglycerate (BPG) can be visualized by a peroxidase reaction, which at the same time visualizes an in situ hybridization (ISH) signal, specific for the X, Y or 21 chromosome. In order to prove that the BPG-positive cells were erythroid, an anti-glycophorin A (GPA) antiserum combined with a staphylococcal rosette technique was used. To enrich for erythroblasts, leukocytes were depleted from maternal blood by treatment with anti-CD45 monoclonal antibody and passage over an anti-mouse IgG-coated glass bead column. To evaluate the potential of the method for clinical use, we studied maternal blood samples from 18 women referred to us for prenatal diagnosis between 6 and 19 weeks of gestation. Erythroblasts were found in 13 out of 14 normal pregnancies. Erythroblasts with a Y-signal were found as early as 9 weeks of gestation, but at 6 weeks the Y-signal was seen in BPG-negative cells only. These cells showed an epithelioid morphology indicating that they were cytotrophoblasts. The BPG-ISH method provides a simple technique for identifying erythroblasts and simultaneously visualizing a desired probe.