Molecular analysis of the rhd genotype in fetuses at risk for rhd hemolytic disease

Abstract

The objective of this study was to evaluate the accuracy of a DNA-based testing methodology in determining the RhD genotypes of fetuses at risk for RhD hemolytic disease. We designed a multiplex polymerase chain reaction-based test based on recent RhD and RhCE sequence information. To improve the accuracy of the results, two different portions of the RhD gene were examined. Deoxyribonucleic acid was extracted from fetal specimens, portions of the RhD gene were amplified by the polymerase chain reaction, and the amplified product was run on a polyacrylamide gel to look for the presence or absence of the RhD gene. We tested 67 amniotic fluid and two chorionic villus specimens to determine the fetal RhD genotype in pregnancies at risk for RhD hemolytic disease. Forty-seven of the 69 specimens were determined to be Rh-positive, and 22 were Rh-negative. Fifty of the 69 fetal specimens--31 Rh-positive and 19 Rh-negative--were serotyped at birth. In all 50, there was complete correlation between the DNA analysis and the serotyping results. RhD gene analysis is a rapid and reliable method that provides an accurate fetal genotype to aid in the prenatal care of RhD-alloimmunized women.

Specificity and sensitivity of RHD genotyping methods by PCR‐based DNA amplification

“…The frequency of false-negative (and false-positive, see above) events should be considerably reduced (or avoided) if at least two pairs of primers probing different regions of the RHD gene are used in routine analysis. Such an approach has been recently considered (Spence et al, 1995;Pope et al, 1995), but without a documented comparison of the different systems. The policy in our Centre is to simultaneously use all four typing methods separately, performed by two different investigators, each exploring only two methods.…”

RhD status of a fetus at risk for haemolytic disease with a discrepant maternal DNA-based RhD genotype

“…For example, a DNA-based genotyping error can have serious consequences to the fetus if recommendations were made to withdraw therapy (Lighten et al, 1995). Therefore, it has been suggested that RhD genotyping of the fetus be performed by evaluating different regions of the gene to avoid genotyping errors (Aubin et al, 1997;Lighten et al, 1995;Pope et al, 1995;Simsek et al, 1994Simsek et al, , 1995Spence et al, 1995).…”

Fetal Rhesus D genotyping on amniocytes in alloimmunised pregnancies using fluorescence duplex polymerase chain reaction

“…To date, most experiences have been obtained using PCR on amniocytes. Previous studies 12–14 showed a nearly perfect correlation between the results of fetal RhD geno‐ and serotyping in a total of 237 pregnancies (error rates 0–1.4%).…”

“…For clinical application, we therefore analysed amniotic fluid specimens in two separate duplex PCR assays each detecting a different RhD gene region (exon 7 and exon 10). Spence et al 14 and Pope et al 23 preferred multiplex PCR, simultaneously amplifying RhD specific DNA fragments at exon 4 through 5 and exon 10. In contrast to these and other study groups, we used fluorescence PCR and automated DNA fragment analysis allowing rapid and objective testing.…”