Determination of the origin of single nucleated cells in maternal circulation by means of random PCR and a set of length polymorphisms.
Non-invasive prenatal diagnosis on fetal nucleated erythrocytes from the maternal circulation is hampered by the small number of nucleated erythrocytes and the uncertainty as to whether they are of fetal or maternal origin. To overcome the latter limitation, single nucleated erythrocytes were separated and enriched from maternal blood by a triple density gradient and a monoclonal antibody (CD71) in combination with a magnetic activated cell sorter. Single nucleated cells were microscopically examined, individually collected with extended Pasteur pipettes, and each transferred into separate caps for the polymerase chain reaction (PCR). The DNA of the single nucleated erythrocytes was amplified at least 50-fold with a random PCR technique, viz., primer extension preamplification. Precise differentiation between maternal and fetal nucleated erythrocytes was achieved via PCR by using primers flanking highly polymorphic nucleotide repeats (DIS53, ACTBP2 and D21S11) and with a XY-specific primer pair (amelogenin). A total of 134 putative nucleated erythrocytes were analyzed from blood samples of 19 pregnant women. With the help of the polymorphic repeats, 25% were assigned as being of maternal origin, 26% of fetal origin, and 48% were uninformative. In cases with male fetuses, the amelogenin primers revealed 30% of cells to be fetal nucleated erythrocytes, the remaining 70% being of maternal origin. The results indicate that the combination of random PCR and PCR-mediated polymorphism analysis on the DNA of single nucleated erythrocytes is a useful technique for non-invasive prenatal diagnosis.
von Eggeling
F
F
Institut für Humangenetik und Anthropologie, Klinikum der FSU Jena, Germany. fegg@mti-n.uni-jena.de
Michel
S
S
Günther
M
M
Schimmel
B
B
Claussen
U
U
D053523:Amelogenin; D001800:Blood Specimen Collection / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003746:Dental Enamel Proteins / Q000235:genetics; D004900:Erythroblasts / Q000166:cytology*; D005260:Female; D005312:Fetal Blood; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D012732:Sex Determination Analysis
D053523:Amelogenin; D003746:Dental Enamel Proteins