Direct quantification of fetal cells in maternal blood by real-time PCR.
BACKGROUND
Fetal cells in maternal blood still present an enticing alternative for the development of a safe and efficacious non-invasive method for prenatal diagnosis. However, most enrichment methods are very tedious and have failed to realise this long sought after goal. We developed a simple, robust TaqMan real-time PCR assay to directly quantify male fetal cells in maternal blood using the multi-copy DYS14, without the need for any additional enrichment procedure.


METHODS
The sensitivity of the DYS14 assay was evaluated by using female genomic DNA spiked with male DNA or male cells. The specificity of the DYS14 assay was evaluated by examining 40 adult blood samples and 44 maternal blood samples from pregnant women with known fetal gender. Direct quantification of fetal cells in maternal blood was performed in 14 of the maternal blood samples by the DYS14 assay. The results were compared to those by SRY assay.


RESULTS
The sensitivity of DYS14 PCR assay was found to be higher than that of SRY assay for the detection of fetal cells. The number of fetal cells in maternal blood did not exceed 2/mL blood. The presence of cell-free fetal DNA in maternal blood could lead to erroneous quantification of fetal cell DNA. The number of fetal cells detected in blood cell pellets, which had been unwashed to remove the cell-free fetal DNA, was indeed significantly higher than those in extensively washed samples.


CONCLUSIONS
We quantified the fetal cells in maternal blood without fetal cell enrichment procedures by TaqMan real-time PCR for a multi-copy DYS14 locus. Our assay is sensitive and also suitable for automation, and may be a useful tool for the determination of fetal-maternal cell trafficking, such as microchimerism.
Zhong
Xiao Yan
XY
Laboratory for Prenatal Medicine and Experimental Oncology, Department of Research, University Women's Hospital Basel, Basel, Switzerland. xzhong@uhbs.ch
Holzgreve
Wolfgang
W
Hahn
Sinuhe
S
D041322:Chromosomes, Human, Y; D004247:DNA / Q000097:blood*; D005260:Female; D005333:Fetus; D006801:Humans; D008297:Male; D008431:Maternal-Fetal Exchange; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D011262:Pregnancy Trimester, Second; D011296:Prenatal Diagnosis / Q000379:methods; D012680:Sensitivity and Specificity; D012732:Sex Determination Analysis / Q000379:methods*
D004247:DNA