Detection of fetal messenger ribonucleic acid in maternal blood to determine fetal RhD status as a strategy for noninvasive prenatal diagnosis,☆☆,,★★

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Abstract

OBJECTIVES: Our purpose was to test the hypothesis that reverse transcriptase–polymerase chain reaction for fetal messenger ribonucleic acid in maternal blood is more sensitive than polymerase chain reaction from genomic deoxyribonucleic acid in prenatal determination of fetal RhD blood type.

STUDY DESIGN: Fetal nucleated erythrocytes in peripheral blood from 35 RhD-negative women were enriched by triple-density gradient centrifugation, anti-CD71 magnetic sorted, and deoxyribonucleic acid and ribonucleic acid extracted. Sensitivities of reverse transcriptase–polymerase chain reaction and polymerase chain reaction were compared to predict definitive fetal RhD blood type determined in fetal tissues.

RESULTS: Reverse transcriptase–polymerase chain reaction was significantly more accurate (P = .03) than genomic–polymerase chain reaction in predicting fetal RhD blood type, both overall (28 of 35 vs 22 of 35) and when the fetus was RhD-positive (12 of 19 vs 6 of 19).

CONCLUSIONS: Reverse transcriptase–polymerase chain reaction is more sensitive than genomic–polymerase chain reaction in detection of fetal RhD sequences in maternal blood. (Am J Obstet Gynecol 1998;179:210-4.)

Section snippets

Sample collection

Institutional ethical committee approval was obtained for this study, which complied with national guidelines relating to research in fetal tissues. Three different groups of RhD-negative women were investigated: women in the first trimester at 6 to 12 weeks’ gestation undergoing termination of pregnancy, women in the second trimester (13 to 23 weeks’ gestation) undergoing genetic amniocentesis, and women in the third trimester at ≥36 weeks’ gestation. None received anti-D antenatally. Venous

Results

Reverse transcriptase–PCR generated an amplification product of 567 bp. PCR for DNA generated an amplification product of 286 bp. Thirty-five RhD-negative pregnant women were included in the study; 10 in the first trimester, 10 in the second trimester, and 15 in the third trimester. Table I shows the comparison between reverse transcriptase–PCR and genomic PCR in each of the gestational groups.

. Accuracy of reverse transcriptase–PCR and genomic PCR by trimester in prenatal prediction of fetal

Comment

This is the first study to compare reverse transcriptase–PCR with genomic PCR in the same patient group. We found that reverse transcriptase–PCR for detection of fetal RhD messenger RNA in the maternal circulation is more sensitive than PCR for detection of fetal DNA. This is presumably due to a greater template copy number. Although the copy number for RhD is currently unknown, it is likely (as for other expressed sequences) to be many hundred-fold greater than the single strand of RhD DNA

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From the Institute of Obstetrics and Gynaecology, Imperial College School of Medicine, Queen Charlotte’s and Chelsea Hospital,a and the Universitats Frauenklinik, Kantonspital.b

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Supported by a North Atlantic Treaty Organization Collaborative Research Grant, an Eden Traveling Fellowship from the Royal College of Obstetricians and Gynaecologists, and a grant from the Swiss National Foundation.

Reprints not available from the authors.

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0002-9378/98 $5.00 + 0   6/1/88955

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