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 present in maternal blood have been reported. Here we describe the use of a quantitative fluorescence in situ hybridization (FISH) method and in vitro suspension cultures of erythroid cells from newborn cord blood and maternal peripheral blood. Together with a rapid high performance liquid chromatography (HPLC) method, that allows us to determine as few as 100 cells containing haemoglobin F (HbF), we have scrutinized the reported enrichment methods for fetal nucleated cells in peripheral maternal blood. One hundred FISH analyses on maternal peripheral blood were performed. The method comprises a cell lysis method for depletion of red cells with minimal losses of nucleated cells, uniform numbers of cells (750 000 cells each) on microscopic slides, and inclusion of internal controls to monitor the efficacy of hybridization. Twenty-six cultures of pure erythroid progenitor cells from maternal peripheral blood were analysed for the expansion of fetal cells. To generate these in vitro cultures, nucleated cells from 10-20 ml of peripheral blood 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 8x10(8) maternal cells were added into tissue culture medium for these 13 cultures, resulting in about 2x10(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 quantitatively, we could not find any fetal cells in cultures from maternal blood. Likewise, in 7.5x10(7) peripheral blood cells probed by FISH analysis (half of which were from pregnancies with male fetuses) no single Y chromosome was detected. In summary, suspension cultures of erythroid cells can be established 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 current technology.
Huber
K
K
Ludwig Boltzmann Inst. f. molekulargenetische Laboratoriumsdiagnostik, Donauspital SMZ-Ost, Vienna, Austria.
Bittner
J
J
Worofka
B
B
Rosen
A
A
Hafner
E
E
Philipp
K
K
Bauer
K
K
D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D015672:Erythroid Precursor Cells / Q000166:cytology*; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005319:Fetal Hemoglobin / Q000032:analysis; D005434:Flow Cytometry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D016133:Polymerase Chain Reaction; D011247:Pregnancy
D005319:Fetal Hemoglobin