K. Huber et al., Quantitative FISH analysis and in vitro suspension cultures of erythroid cells from maternal peripheral blood for the isolation of fetal cells, PRENAT DIAG, 20(6), 2000, pp. 479-486
Citations number
20
Categorie Soggetti
Reproductive Medicine","Medical Research Diagnosis & Treatment
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.