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

Frederik Banch Clausen; Grethe Risum Krog; Klaus Rieneck; Emma Elin Frida Råsmark; Morten Hanefeld Dziegiel

doi:10.1159/000321347

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

Frederik Banch Clausen, Grethe Risum Krog, Klaus Rieneck, Emma Elin Frida Råsmark, Morten Hanefeld Dziegiel

16 Citationer (Scopus)

Abstract

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.

Originalsprog	Engelsk
Tidsskrift	Fetal Diagnosis and Therapy
Vol/bind	29
Udgave nummer	2
Sider (fra-til)	155-63
Antal sider	9
ISSN	1015-3837
DOI	https://doi.org/10.1159/000321347
Status	Udgivet - mar. 2011

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10.1159/000321347

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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 et al.

I: Fetal Diagnosis and Therapy, Bind 29, Nr. 2, 03.2011, s. 155-63.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

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abstract = "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.",

keywords = "Female, Fetal Diseases, Humans, Mass Screening, Polymerase Chain Reaction, Pregnancy, Rh Isoimmunization, Rh-Hr Blood-Group System, Sensitivity and Specificity",

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AU - Clausen, Frederik Banch

AU - Krog, Grethe Risum

AU - Rieneck, Klaus

AU - Råsmark, Emma Elin Frida

AU - Dziegiel, Morten Hanefeld

PY - 2011/3

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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.

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KW - Humans

KW - Mass Screening

KW - Polymerase Chain Reaction

KW - Pregnancy

KW - Rh Isoimmunization

KW - Rh-Hr Blood-Group System

KW - Sensitivity and Specificity

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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

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