Quantitative FISH analysis and in vitro suspension cultures of erythroid cells from maternal peripheral blood for the isolation of fetal cells

Several techniques for the enrichment of nucleated fetal red blood cells pr esent in maternal blood have been reported. Here we describe the use of a q uantitative fluorescence in situ hybridization (FISH) method and in vitro s uspension cultures of erythroid cells from newborn cord blood and maternal peripheral blood. Together with a rapid high performance liquid chromatogra phy (HPLC) method, that allows us to determine as few as 100 cells containi ng haemoglobin F (HbF), we have scrutinized the reported enrichment methods for fetal nucleated cells in peripheral maternal blood. One hundred FISH a nalyses on maternal peripheral blood were performed. The method comprises a cell lysis method for depletion of red cells with minimal losses of nuclea ted cells, uniform numbers of cells (750 000 cells each) on microscopic sli des, and inclusion of internal controls to monitor the efficacy of hybridiz ation. Twenty-six cultures of pure erythroid progenitor cells from maternal peripheral blood were analysed for the expansion of fetal cells. To genera te these in vitro cultures, nucleated cells from 10-20 ml of peripheral blo od from 26 pregnant women were grown in media containing growth factors and hormones to yield over 10(7) of immature erythroid cells within two weeks. Of those, 13 cultures were from pregnancies with confirmed male fetuses. A total of approximately 8 x 10(8) maternal cells were added into tissue cul ture medium for these 13 cultures, resulting in about 2 x 10(8) nearly pure erythroid cells after two weeks. Whereas fetal cells, alone or added into cultures of peripheral blood, grow rapidly and can be detected quantitative ly, we could not find any fetal cells in cultures from maternal blood. Like wise, in 7.5 x 10(7) peripheral blood cells probed by FISH analysis (half o f which were from pregnancies with male fetuses) no single Y chromosome was detected. In summary, suspension cultures of erythroid cells can be establ ished routinely and easily. With the quantitative FISH technique used, 750 000 cells per slide can be screened reliably for cells with Y chromosomes. However, the stringent quality-criteria and most elaborate methods indicate that fetal cells in maternal peripheral blood can not be found using the c urrent technology. Copyright (C) 2000 John Wiley & Sons, Ltd.