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
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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. / Clausen, Frederik Banch; Krog, Grethe Risum; Rieneck, Klaus; Råsmark, Emma Elin Frida; Dziegiel, Morten Hanefeld.
In: Fetal Diagnosis and Therapy, Vol. 29, No. 2, 01.01.2011, p. 155-63.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - 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
AU - Clausen, Frederik Banch
AU - Krog, Grethe Risum
AU - Rieneck, Klaus
AU - Råsmark, Emma Elin Frida
AU - Dziegiel, Morten Hanefeld
N1 - Copyright © 2010 S. Karger AG, Basel.
PY - 2011/1/1
Y1 - 2011/1/1
N2 - 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.CONCLUSION: 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.
AB - 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.CONCLUSION: 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.
KW - Female
KW - Fetal Diseases
KW - Humans
KW - Mass Screening
KW - Polymerase Chain Reaction
KW - Pregnancy
KW - Rh Isoimmunization
KW - Rh-Hr Blood-Group System
KW - Sensitivity and Specificity
U2 - 10.1159/000321347
DO - 10.1159/000321347
M3 - Journal article
C2 - 21071922
VL - 29
SP - 155
EP - 163
JO - Fetal Diagnosis and Therapy
JF - Fetal Diagnosis and Therapy
SN - 1015-3837
IS - 2
ER -
ID: 34143971