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

Abstract

Summary.We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I-III were the most sensitive and gave a detectable signal with D-pos/D-neg mixtures containing only 0 . 001% D-positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10-50 times less. Investigation of D variants indicated that false-negative results were obtained with method II (D IVb variant), method III (D VI and DFR variants) and method IV (D VI variants), but not method I. Weak D (D u ) was correctly detected as D-positive by all methods, but most cases of Rh null appeared as false-positives, as they carry normal RH genes that are not phenotypically expressed. Some false-positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD-neg but carrying a C-or E-allele, whereas a high incidence of false-positives was found among non-Caucasian Rh-negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.

Distribution of Rhesus Blood Group System in the French Basques: A Reappraisal Using the Allele-Specific Primers PCR Method

“…These data were obtained using serologic typing and one could speculate Touinssi/Chiaroni/Degioanni/De Micco/ Dutour/Bauduer on a possible lack of accuracy of this technique in the molecular biology era. In this paper, we report the distribution of Rh polymorphism within a sample of autochthonous French Basques using for the first time an adapted PCR technique that avoids theoretically mistyping errors sometimes encountered with serology [12,13]. In addition, we compare our results with those of published series from various Basque subpopulations.…”

Noninvasive prenatal diagnosis of fetal blood group phenotypes: current practice and future prospects

“…Where possible, RHD genes producing variant D antigens should give a positive result and this can be achieved by including an amplification of exon 7 or 10. Many laboratories prefer to include an amplification of exon 7, which appears to provide a higher affinity reaction than exon 10 (Aubin et al, 1997;Rouillac-Le Sciellour et al, 2004). Those methods that just employ amplification of exon 7 and 10 are not suitable for testing any population containing people of African origin, as they will give false-positive results when the fetus has RHD .…”

D^Va Category Phenotype and Genotype in Japanese Families

“…With SSP–PCR, we tested the nucleotide position (nt) 744 on exon 5 of the RHD gene (D5b), and nt 985, 992 and 1048 on exon 7 (D7a, D7b), as previously described [3]. Additionally, we used primers [4]testing nt 514 and 602 on exon 4 and applied the same PCR protocol as previously published for exons 2, 5 and 7 [3]. The D–specific reaction D5b (exon 5 of the RHD gene) was repeated without primers for the internal control and the amplification products were incubated with restriction enzymes Hin c II (Promega, Madison, Wisc., USA), Taq I (Promega) and Nla III (New England Biolabs, Schwalbach, Germany), testing the D–specific nt 667, 697 and 712, respectively.…”