The use of polymerase chain reaction to determine fetal RhD status

Center for Medical Genetics, Johns Hopkins University School of Medicine, Baltimore, MD.
American Journal of Obstetrics and Gynecology (Impact Factor: 4.7). 11/1994; 171(4):1047-51. DOI: 10.1016/0002-9378(94)90032-9
Source: PubMed


Our purpose was (1) to establish the accuracy of a deoxyribonucleic acid amplification method in determination of RhD status in adult blood samples, including weak D variants (previously referred to as Du) and a D mosaic, and (2) to apply the method to determine fetal RhD status in alloimmunized pregnancies.
Twenty-five adult blood samples, including five weak D variants and one D mosaic, were analyzed with a polymerase chain reaction to determine RhD type. The method was then applied to amniotic fluid samples obtained by amniocentesis from three RhD-negative women with known RhD sensitization.
RhD type determined by polymerase chain reaction for all adult blood samples agreed with serologic typing results. All weak D variants and the D mosaic gave results consistent with RhD positivity. Fetal RhD status was determined in each of the three alloimmunized pregnancies, and obstetric management decisions were made on the basis of these results.
This polymerase chain reaction method allows rapid and accurate determinations of fetal RhD status by amniocentesis. Fetal blood sampling or serial amniocenteses may be avoided when the fetus is RhD negative, and plans for surveillance and intervention can be confidently made if the fetus is RhD positive. However, before the widespread use of this assay, its sensitivity and specificity must be established. Because weak D variants and a D mosaic demonstrated RhD-positive status by polymerase chain reaction, the method described is applicable to these RhD variants.

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    ABSTRACT: To evaluate the accuracy of polymerase chain reaction (PCR) amplification of a portion of the RhD gene by testing a large number of DNA samples derived from individuals whose RhD status was established by the standard serologic method. Seven hundred sixty-five samples were obtained from two sources: subjects taking part in studies at the University of Iowa Hospitals and Clinics (n = 107), and Centre d'Etude du Polymorphisme Humain (CEPH) families used for studies of genetic variation (n = 658). Deoxyribonucleic acid was extracted from blood samples of University of Iowa volunteers and from CEPH families by standard techniques. With few modifications, published primers, reaction and electrophoresis conditions, which yield a 1200-bp fragment in all individuals and a 600-bp fragment in RhD-positive individuals, were used. By standard serologic techniques, we identified samples from 632 RhD-positive and 133 RhD-negative individuals. Two (both from CEPH) of the 632 RhD-positive individuals were characterized as RhD-negative by PCR. Seven of the 133 RhD-negative samples were judged to be RhD-positive by PCR because of the presence of a light 600-bp band. Despite repeated attempts, no bands or DNA were identified in one RhD-negative sample. Thus, the sensitivity of RhD typing by PCR was 99.7%, the specificity 94.0%. Based on our data, it would appear that the use of PCR to establish RhD type can be introduced cautiously into current management schemes in the evaluation of RhD sensitization.
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