Non-invasive detection of fetal rhesus D status: a comparison between polymerase chain reaction and flow cytometry.
OBJECTIVE
A non-invasive prenatal determination of the fetal RhD status might be useful for the management of pregnancies in RhD-negative women whose partners are RhD positive.


METHODS
Maternal peripheral blood of 32 RhD-negative women (17-24 weeks of gestation) was collected, and circulating fetal cells were enriched by CD71 mini-magnetic activated cell sorting. The RhD status of the fetuses was assessed using multiparametric flow cytometry, and results were compared to those of reverse transcriptase (RT)-polymerase chain reaction (PCR), or PCR, which acted as control. Flow-cytometric study of fetal cells employed monoclonal antibodies directed against CD71, glycophorin A (GPA) and RhD antigens.


RESULTS
The median percentage of CD71- and RhD-positive cells was 0.83% (range 0.14-6.44%), and that of CD71 and GPA-positive cells was 10.07% (range 0.52-45.84%). Flow-cytometric analysis correlated with RT-PCR results of RNA obtained from whole maternal blood. In 1 case, an incorrect result was due to the failure of the amplification of the specific RhD band on RNA extracted from the CD71-positive fraction. In two instances, we observed false-positive results for RhD in PCR of DNA obtained from maternal plasma.


CONCLUSIONS
Based on our results, flow-cytometric analysis might be proposed as a clinical tool for the non-invasive prenatal determination of the fetal RhD status independently of fetal gender.
Di Simone
Nicoletta
N
Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Rome, Italy.
Lai
Marco
M
Rumi
Carlo
C
Riccardi
Patrizia
P
D'Asta
Marco
M
Leone
Giuseppe
G
Mancuso
Salvatore
S
Caruso
Alessandro
A
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000032:analysis; D001788:Blood Grouping and Crossmatching / Q000379:methods*; D004900:Erythroblasts / Q000737:chemistry; D005260:Female; D005312:Fetal Blood / Q000166:cytology; D005434:Flow Cytometry; D005865:Gestational Age; D006021:Glycophorins / Q000032:analysis; D006801:Humans; D011247:Pregnancy; D011296:Prenatal Diagnosis; D011990:Receptors, Transferrin / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012204:Rh-Hr Blood-Group System / Q000097:blood* / Q000145:classification / Q000235:genetics
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C068126:CD71 antigen; D006021:Glycophorins; D011990:Receptors, Transferrin; D012204:Rh-Hr Blood-Group System