P25.15: Magnetic cell sorting and flow cytometry for the detection of fetal nucleated erythroblasts in maternal circulation during the first trimester of pregnancy

Abstract

The prenatal diagnosis of isolated unilateral reduction of the fetal upper limb is one of the most difficult problems. In our experience fetal extremities can be evaluated more easily in early pregnancy (between 12-14 weeks) by transvaginal sonography than later in pregnancy. P25.15Magnetic cell sorting and flow cytometry for the detection of fetal nucleated erythroblasts in maternal circulation during the first trimester of pregnancy We describe two methods for isolation of nucleated fetal red blood cells from peripheral maternal blood. We used magnetic cell sorting (MACS) and flow cytometry in order to isolate the fetal erythroblasts from 20 ml maternal peripheral blood. Method: In the first group of the study participated twenty pregnant women who were between 8 and 14 weeks of gestation (mean 10.8), and had low risk for Down syndrome according to their age (between 21 to 34 years old and mean 28.1). After removal of the mononuclear cell layer we have performed magnetic cell sorting twice by using the glycophorin A antibody and secondary antibody conjugated with magnetic beads. The positive cells have been held on the magnet and were maternal and fetal red cells. We have then added on the positive cells the monoclonal antibody for γ-chain and the fetal nucleated erythrocytes have been measured under light microscope. The second group in our study consisted of 15 pregnant women between 8 and 14 weeks of gestation and we performed flow cytometry. After removal of the mononuclear cell layer we have added the antibodies CD 45 ECD, CD 41 PE, GlycA PE and anti γ-FITC and performed flow cytometry. The isolated population of cells was CD45-, CD41-, GlycA+, IGγ+ and contains the nucleated red blood fetal cells. Results:The number of fetal erythrocytes we have counted with MACS was between 0 and 18, with mean number of 7.2, standard deviation (SD) 5.2 and standard error of mean (SEM) 1.17. We have managed to find these cells on the 18 out of 20 cases (90%). The mean number of the isolated cells with flow cytometry were 1138 (363-2560), SD 631.6 and SEM 163. The percentage of the isolated cells to the total cell population was 0.8% to 4.2% (mean 1.7%, SD 0.6) The aim of our study was to identify the nucleated red fetal blood cells in maternal blood. We have identified all the obstacles encountered in our experiments and we have finally managed to isolate few nucleated fetal red blood cells from the maternal circulation in most samples. P25.16What type of management should we choose with positive combined test and normal karyotype in the first trimester? D. Smetanova, E. Kulovany, B. Kubesova, M. Hynek Fetal Medicine, GENNET, Praha, Czech RepublicObjectives: To assess optimal pregnancy management in the case of first trimester combined test positivity and normal karyotype. Methods: Between 2004 and 2007 overall 9 524 first trimester combined tests were carried out and at 180 (1.8%). CVS was done due to combined risk of chromosomal aberration or presence of US fetal malformation. US scans were provided by FMF certified so...