TY - JOUR
T1 - Evaluation of prenatal RHD typing strategies on cell-free fetal DNA from maternal plasma
AU - Grootkerk-Tax, Martine G. H. M.
AU - Soussan, Aicha Ait
AU - de Haas, Masja
AU - Maaskant-van Wijk, Petra A.
AU - van der Schoot, C. Ellen
PY - 2006
Y1 - 2006
N2 - 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 RHDpsi mothers and that RHDpsi 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 RHDpsi, 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-RHDpsi+ 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 RHDpsi+ 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
AB - 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 RHDpsi mothers and that RHDpsi 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 RHDpsi, 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-RHDpsi+ 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 RHDpsi+ 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
U2 - https://doi.org/10.1111/j.1537-2995.2006.01044.x
DO - https://doi.org/10.1111/j.1537-2995.2006.01044.x
M3 - Article
C2 - 17176327
SN - 0041-1132
VL - 46
SP - 2142
EP - 2148
JO - Transfusion
JF - Transfusion
IS - 12
ER -