Determination of fetal RHD type in plasma of RhD negative pregnant women

Abstract

Alloimmunization against the RhD antigen is the most common cause of hemolytic disease of the fetus and newborn. Antenatal anti-D prophylaxis in addition to postnatal anti-D prophylaxis reduces the number of RhD-immunizations compared to only postnatal administration. Cell-free fetal DNA released from the apoptotic trophoblastic placental cells into the maternal circulation can be used to determine the fetal RHD type in a blood sample from an RhD negative mother. Based on this typing, antenatal anti-D prophylaxis can be recommended only to RhD negative women carrying an RhD positive fetus, since only these women are at risk of developing anti-D. The objective was to establish and validate a method for non-invasive fetal RHD typing. The fetal RHD genotype was studied in 373 samples from RhD negative pregnant women (median gestational week 24). DNA extracted from plasma was analyzed for the presence/absence of RHD exon 7 and 10 in a real-time PCR. The RHD genotype of the fetus was compared with the serological RhD type of the newborn. In 234 samples, the fetal RHD test was positive and in 127 samples negative. There was one false positive and no false negative results. In 12 samples, the fetal RHD type could not be determined, in all of them due to a maternal RHD gene. This method gives a reliable detection of fetal RHD positivity in plasma from RhD negative pregnant women. Antenatal anti-D prophylaxis based on the predicted fetal RhD type will avoid unnecessary treatment of pregnant women carrying an RhD negative fetus.

Keywords:

• Rho(D) Immune Globulin
• RHO(D) antibody
• blood typing
• fetus
• genotyping
• prenatal diagnosis/methods
• immunization
• pregnancy
• newborn
• DNA
• real-time polymerase chain reaction

Acknowledgements

We thank Frederik B. Clausen (Department of Clinical Immunology, Copenhagen University Hospital, Denmark) for invaluable guidance during the work and the contribution with plasma samples. We are grateful to Håkon Wergeland and Alka Saigal and midwives at the Department of Obstetrics for their great efforts in the recruitment of pregnant women to the study and to midwives at the Health Centers in Oslo for their contribution. Last we thank Åsa Hellberg (Nordic Reference Laboratory of Blood Group Genomic Typing, Clinical Immunology and Transfusion Medicine, Lund, Sweden) for the molecular biology investigations.

Disclosure statement

No potential conflict of interest was reported by the authors.