The Research Of Cell-Free Fetal DNA In Maternal Blood In Predicting Fetal Blood Groups

Background:Hemolytic disease of the newborn （HDN） is the hemolytic disease,due to the mismatchof red blood cell blood groups between mothers and fetus.The literatures reported that the red blood groupscausing HDN included the ABO,Rh,MNSs,Kell,Kidd,Yt,Duffy and Diego.However,the most commoncause is the mismatch of ABO and RhD between the mothers and fetus.The maternal IgG antibodies canthrough the placenta and combine with corresponding blood group antigens on the surface of red bloodcell, resulting in the immunological destruction of fetal red blood cell. It is of positively clinicalsignificance to the prediction of the incidence of HDN that prenatal detection of fetal ABO and RhD bloodgroups. The methods of prenatal prediction of fetal blood groups can be divided into two types, includingthe invasive and the non-invasive. The invasive methods,such as the centrifugation of amniotic fluidcells,the biopsy of placenta tissue,cordocentesis,chorionic villus sampling and amniocentesis,should beapplicated prudently in clinical practice,due to the risk to the mothers and fetus,such as transplacentalhaemorrhage and spontaneous abortion. In the non-invasive methods, all sorts of detection can be mademainly through the isolation of intact fetal cells from the maternal circulation by flow cytometry.However,these methods need expensive equipments and operators of higher technical requirements.In1997,Lo and his cooperaters found that cell-free fetal DNA could be detected in the maternalcirculation during pregnancy.There are three kinds of the most possible sources of the fetal DNA in thematernal plasma, including the destruction of fetal cells in maternal blood, transplacental transfer of fetalcell-free DNA, and the destruction of villous trophoblasts bordering the intervillous spaces.Theconcentration of fetal free DNA is associated with gestational age, and can be detected as early as in the fourth gestational age. Furthermore, there is an increase in fetal DNA concentration as gestation advanced,with a remarkable sharply increase during the last8weeks of pregnancy.Moreover, except for the regularlychanges during the whole pregnancy, the concentration of fetal DNA would increase significantly duringsome fetal disease state compared with normal levels.Therefore, the fetal DNA can be used to prenataldiagnosis some fetal genetic diseases. In this research, we use the cell-free fetal DNA in maternal blood toprenatally predict fetal ABO and RhD blood groups,based on the molecular basis of the human ABO andRhD blood groups.It is a novel and non-invasive method,which can avoid the trauma to maternal and therisk of spontaneous abortion.Objective The fetal free DNA in the maternal circulation is used to prenatally detect fetal ABO andRhD blood groups,and to prevent the occurrence of hemolytic disease of newborn.Methods (1)90samples were collected from the pregnant women whose ABO blood group is O andtheir spouses randomly.we detected the ABO blood group by serological method,used2-mercaptoethnol toneutralize the IgM anti-A and anti-B antibodies,and used double dilution method to detect the titer of IgGanti-A and anti-B in the maternal blood.We also extracted the fetal free DNA, and verified it by detectingthe SRY gene.We selected the samples which were positive to SRY, and then genotyped the fetal ABOblood group by Polymerase Chain Reaction-Restriction fragment length polymorphism (PCR-RFLP)prenatally.Finally,we detected the fetal ABO blood group by serological method postnatally,and comparedwith results by using the fetal DNA prenatally.(2)80samples were collected from the RhD negativepregnant women who had previons pregnant history and their spouses.We detected their RhD blood groupby serological method,detected the D antigen on the red blood cells by indirect antiglobulin test andadsorption-elution techniques. We also extracted the fetal free DNA and verified it by detecting the SRYgene.We selected the samples which were positive to SRY, and then genotyped the fetal ABO blood group by Polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP) prenatally, Finally, we detected thefetal RhD blood group by serological method postnatally, and compared with results by using the fetalDNA prenatally.Results In the90samples collected from the pregnant women whose ABO blood group is O,thepositive rate of SRY gene is52.22%(47/90).However,in the80samples collected from the pregnant womenwho were RhD negative, the positive rate of SRY gene is52.50%(42/80).Compared the fetal blood groupprenatally predicted by the fetal DNA with the fetal blood group postnatally detected by the serologicalmethods,the results showed that the coincidence rates of these blood groups were different significantly.Inthe ABO blood group,the coincidence rate of A was84.21%(16/19), the coincidence rate of B was81.25%(13/16),and the coincidence rate of O was75.00%(9/12); In the RhD blood group,the coincidencerate of the RhD positive was89.29%(25/28),while the coincidence rate of the RhD negative was78.57%(11/14).Conclusion Fetal cell-free DNA can be detected in the maternal circulation during pregnancy.It isfeasible to use the fetal cell-free DNA to predict the fetal ABO and RhD blood groups prenatally.