Evaluation of prenatal RHD typing strategies on cell-free fetal DNA from maternal plasma

• Published in: Transfusion (Philadelphia, Pa.) Pa.), 2006-12, Vol.46 (12), p.2142-2148
• Main Authors: Grootkerk-Tax, Martine G.H.M., Soussan, Aicha Ait, De Haas, Masja, Maaskant-van Wijk, Petra A., Van Der Schoot, C. Ellen
• Format: Article
• Language: English
• Subjects: Anesthesia. Intensive care medicine. Transfusions. Cell therapy and gene therapy; Biological and medical sciences; Blood Grouping and Crossmatching - methods; Blood. Blood and plasma substitutes. Blood products. Blood cells. Blood typing. Plasmapheresis. Apheresis; Bone marrow, stem cells transplantation. Graft versus host reaction; DNA - blood; Exons; Female; Fetal Blood - immunology; Genotype; Hematology; Humans; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Polymerase Chain Reaction; Pregnancy - blood; Rh-Hr Blood-Group System - genetics; Transfusions. Complications. Transfusion reactions. Cell and gene therapy
• ISSN: 0041-1132; 1537-2995
• DOI: 10.1111/j.1537-2995.2006.01044.x

BACKGROUND: The discovery of cell‐free fetal DNA in maternal plasma led to the development of assays to predict the fetal D status with RHD‐specific sequences. Few assays are designed in such a way that the fetus can be typed in RHDψ mothers and that RHDψ fetuses are correctly typed. Owing to the limited knowledge about the mechanism responsible for the presence of fetal DNA in maternal plasma, precautions in developing prenatal genotyping strategies must be made. STUDY DESIGN AND METHODS: Real‐time quantitative (RQ)‐polymerase chain reaction (PCR) assays were developed for prenatal diagnostic use with cell‐free fetal DNA from maternal plasma. An RQ‐PCR assay on RHD exon 5 (amplicon 361 bp), negative on RHDψ, was developed with genomic DNA and evaluated with cell‐free fetal DNA. A previously published RHD exon 5 RQ‐PCR (amplicon 82 bp) was duplexed with an in‐house developed RHD exon 7 RQ‐PCR and evaluated with cell‐free fetal DNA from pregnant D–RHDψ+ women. RESULTS: The RHD exon 5 361 bp assay showed on cell‐free plasma DNA from D– women carrying a D+ fetus, low amplification levels, resulting in high Ct values and false‐negative results. Owing to fragmentation of cell‐free plasma DNA, too few DNA stretches of sufficient length (>360 bp) are present. The RHD exon 5 82 bp and exon 7 RQ‐PCR duplex was evaluated with RHDψ+ cell‐free plasma DNA and showed complete specificity and maximal sensitivity. CONCLUSION: Assays designed for prenatal genotyping should be developed and evaluated on cell‐free plasma DNA. Prenatal RHD typing is accurate with the RHD exon 5 82 bp and exon 7 duplex strategy.