How do we identify RHD variants using a practical molecular approach?

Abstract

Serologic resolution of Rh discrepancies due to partial D or weak D phenotypes is a frequent problem encountered during routine typing that can be solved by RHD genotyping because it provides better characterization of these variants. The objective of the current study was to develop algorithms for identification of D variants in multiethnic populations based on a logic sequence of molecular tests using a large number of atypical RhD specimens. Thus, a total of 360 blood samples with atypical D antigen expression were analyzed. A previously published multiplex polymerase chain reaction (PCR) procedure was performed and depending on multiplex PCR analysis, the associated RHCE allele, and D variant frequency in our population, an algorithm was developed composed of six flow charts using specific PCR-restriction fragment length polymorphism and/or specific exon sequencing. This strategy allowed the identification of 22 different variants with few assays and a much reduced cost. This study describes a simple and practical algorithm that we use to determine RHD genotypes in samples with unknown RHD. This strategy is relatively easy to implement and the algorithm can be adapted to populations with various ethnic backgrounds after an initial assessment of the type and frequency of D variants. Essentially, we demonstrate that sequencing of all RHD exons is not necessary for the identification of the majority of known D variants.

High frequency of variant RHD genotypes among donors and patients of mixed origin with serologic weak‐D phenotype

“…*The reason for recommending the transfusion of D-positive units for individuals bearing these variants was based on the lack of alloimmunization reports rather than on definite evidences that they are not prone to anti-D development performing the molecular investigation. 11,25 All studies were concordant with respect to the high prevalence of RHD*DAR and to the relatively lower representability of RHD*weak D types 1, 2, and 3 among…”

“…Previous studies have also evaluated the frequency of D variants among Brazilian donors and patients, but with different criteria for performing the molecular investigation . All studies were concordant with respect to the high prevalence of RHD*DAR and to the relatively lower representability of RHD*weak D types 1, 2, and 3 among Brazilian donors with weak‐D phenotype.…”

“…Arnoni et al have previously published an interesting strategy to investigate D variants among Brazilians using the same multiplex PCR which was also the cornerstone of the molecular investigation workflow proposed by the present study. The main differences between the two protocols are that our focused on investigating only individuals with serologic weak‐D phenotype, aiming to determine the best transfusion protocol in a relatively short period of time, while the other protocol is more extensive and focusing on the evaluation of all samples with suspected variant D phenotype.…”

Novel intronic RHD variants identified in serologically D‐negative blood donors

“…This PCR revealed the presence of exon 9 for two samples, a similar gel pattern to Cde s allele found in 15% of RHD-negative Africans [9]. Based on the review literature, it seems to be a good molecular approach to detect common variants among Africans as Cde s , RHDΨ and weak D type 4 and among Europeans as weak D types 1, 2, 3 and 38 [22,37,38]. However, this multiplex PCR did not allow Table 2 In silico prediction of the effect of mutations predicted to affect splicing on splice acceptor or splice donor sites us to identify the type of our four variants (M1, M2, M3 and M4).…”

Weak D Type 4.2.2 (DAR1.2) in an African child: Serology and molecular characterization

“…In conclusion, some partial D variants are likely to be missed by routine serology; for this reason, to increase the accuracy of Rh typing, the development of DNA-based methods for economic and fast large-scale routine use may be a promising future perspective [10,11].…”