Silica Microbeads Capture Fetal Nucleated Red Blood Cells In Maternal Blood For Identification Of Fetal ABO Genotype

Part one:Using silica microbeads for the capture of fetal nucleated red blood cells in maternal peripheral bloodObjective To investigate the feasibility of obtaining fetal nucleated red blood cells from maternal peripheral blood based on the silica microbeads.At the same time,to explore the optimal gestational weeks to capture fetal nucleated red blood cells from maternal peripheral blood.Methods 1.Density gradient centrifugation was used to obtain the mononuclear cells layer from maternal peripheral blood.Then anti-CD147 antibody modified silicamicrobeadswereemployedtocapturetargetcells.Afterthat,CD71+/?-HbF+/DAPI+tri-immunofluorescence was used to identify the captured cells.2.Nucleated red blood cell line was employed to explore the optimal capture conditions with different concentrations of anti-CD147 antibody?1?g/ml,5?g/ml,10?g/ml and 50?g/ml?and different amounts of silica microbeads?1×10⁴/ml,5×10⁴/ml,10×10⁴/ml and 50×10⁴/ml?,as well as different modification method.3.Fetal nucleated red blood cells were captured from maternal peripheral blood using optimal capture method,and the numbers of fetal nucleated red blood cells in maternal blood with different gestational weeks were counted.Results 1.Anti-CD147 antibody modified silica microbeads could be employed to capture fetal nucleated red blood cells in maternal peripheral blood,and those cells could be identified using tri-color immunofluorescence staining.2.10×10⁴/ml silica microbeads with 10?g/ml anti-CD147 antibody has the highest capture efficiency?P<0.05?.Streptavidin modified silica microbeads has higher capture efficiency compared with unmodified microbeads.3.The average number of fetal nucleated red blood cells in maternal peripheral blood range from 12^thh to 20^thh weeks was?53.40±11.15?/ml,and the maximum number of fetal nucleated red blood cells was about 75/ml at the gestational weeks of 17⁺².Conclusion It is feasible to capture fetal nucleated red blood cells in maternal peripheral blood with anti-CD147 antibody modified silica microbeads.The optimal capture time was around 17^thh weeks of gestation.Part two: Silica microbeads capture fetal nucleated red blood cells in maternal blood for identification of fetal ABO genotypeObjective To investigate the practicability of identifying fetal ABO blood group using fetal nucleated red blood cells in maternal peripheral blood captured by the silica microbeads.Methods 1.Anti-CD147 antibody modified silica microbeads were employed to capture fetal nucleated erythrocytes from maternal blood circulation,and the DNA was extracted from these captured cells.Those pregnant women were followed up to deliver,and DNA was extracted from the umbilical venous blood of newborn.Polymerase chain reaction with sequence specific primer was used to detect the ABO genotype of fetus and newborn,respectively.Serological method was used to validate the neonatal ABO blood group.2.For the male fetus with the same ABO blood group as the pregnant woman,the SRY gene was detected to further confirm that the captured cells were fetal cells.Results 1.The accuracy of detecting fetal ABO blood group employing fetal nucleated erythrocytes captured from maternal blood circulation based on the silica microbeads was 86.7%?52/60?.2.For male fetus with the same ABO blood type as the pregnant woman,the SRY gene detection rate was 100%?14/14?.Conclusion The fetal nucleated red blood cells captured by silica microbeads could be used to noninvasive prenatal identify fetal ABO blood group,which further confirm that the method could be employed to noninvasive prenatal test other fetal abnormality.