Evaluation of two real-time multiplex PCR screening assays detecting fetal RHD in plasma from RhD negative women to ascertain the requirement for antenatal RhD prophylaxis.
OBJECTIVE
To evaluate two different multiplex real-time PCR assays detecting fetal RHD for screening of RhD negative women in relation to antenatal RhD prophylaxis.


METHODS
We designed a duplex assay for the detection of RHD exon 7 and 10 and a triplex assay for the detection of RHD exon 7, 10 and 5. We used the same fluorescent dye for the exon 7 and 10 probes to increase sensitivity; exon 5 was VIC labeled. We evaluated possible inhibition of DNA amplification with dilution experiments. We then tested the two multiplex assays with DNA extracted from 97 plasma samples from 38 RhD negative women in gestational weeks 6-37.


RESULTS
Dilution experiments revealed no inhibition of amplification in the multiplex assays. For plasma samples, the duplex assay was significantly more sensitive than the triplex assay (p < 0.0001). For the duplex assay (exon 7/10), accuracy was 99.0%. For the triplex assay (exon 7/10), accuracy was 94.2%. Detection of exon 5 was less reliable.


CONCLUSIONS
The duplex assay using exon 7/10 was the most reliable for prenatal prediction of fetal RhD type as a candidate assay for screening of RhD negative women in relation to antenatal RhD prophylaxis. The triplex assay needs further optimization.
Department of Clinical Immunology, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, Copenhagen, Denmark. Frederik.Banch.Clausen@rh.dk
Frederik Banch
FB
Clausen
Grethe Risum
GR
Krog
Klaus
K
Rieneck
Emma Elin Frida
EE
Råsmark
Morten Hanefeld
MH
Dziegiel
D005260:Female; D005315:Fetal Diseases / Q000517:prevention & control*; D006801:Humans; D008403:Mass Screening; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D012203:Rh Isoimmunization / Q000517:prevention & control*; D012204:Rh-Hr Blood-Group System / Q000097:blood*; D012680:Sensitivity and Specificity
D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen