RHD sequencing: a new tool for decision making on transfusion therapy and provision of Rh prophylaxis

Abstract

The serological differentiation of weak D from partial D, D-negative and D-positive is not always unequivocal. Therefore, sequencing of the RHD gene is required in some cases. Very recently, several new differences between RHD and RHCE have been identified which permitted us to design primers close to the exon/intron boundaries of the RHD-exons. We evaluated these primers in 83 D-positive and 18 D-negative blood donors and applied the new method for the characterization of the RHD gene in six individuals with weak D phenotype. The amplification reactions were concordant with serological findings in 100 of 101 donors (99.0%). In one D-positive donor the PCR for exons 2 and 5 gave a negative result, while the sequence of the remaining eight exons was unchanged. By sequencing samples with very weak D serological reactions, we identified weak D type 4.2.2 and weak D type 15, both previously reported to be associated with anti-D-alloimmunization. Consequently, we recommended the selection of D-negative blood in the weak D type 4.2.2 patient, and the provision of Rh prophylaxis for pregnant women with weak D type 15. In summary, a new RHD sequencing method was developed which can be applied if serological reactions are inconclusive.

Whole exon 5 and intron 5 replaced by RHCE in DVa(Hus)

“…Full-length RHD coding region was sequenced according to a published method (Legler et al 2001). Briefly, ten exons of RHD were amplified separately with ten pairs of RHD-specific primers that target the complementary sequences in introns.…”

Molecular genetics and clinical applications for RH

“…Anti-D immunization in weak D carriers is rare, but there are exceptions: examples include weak D type 15, weak D type 4.2, also known as DAR, and weak D type 7 37–39. The weak D types 1, 2, 3, and 4.0/4.1, which are the most prevalent in any European and Caucasian population, represent more than 95% of all weak D types.…”

Policies and Procedures Related to Testing for Weak D Phenotypes and Administration of Rh Immune Globulin: Results and Recommendations Related to Supplemental Questions in the Comprehensive Transfusion Medicine Survey of the College of American Pathologists

“…Wagner and colleagues 17 proposed an Rh typing and transfusion strategy based on D antigen density that would interpret approximately 95% of all persons of white race/ethnicity with a weak D phenotype as Rh positive. Legler and colleagues 32 proposed an RHD sequencing method for selective application when serological reactions are inconclusive. Flegel 37 proposed managing weak D phenotypes based on the observation that most weak D phenotypes are associated with specific CDE haplotypes and supplementing sensitive gel serological testing with monoclonal reagents and selective RHD genotyping.…”

“…17,27,32,[36][37][38][39] Investigators have proposed algorithms based on RHD genotyping as alternatives to the practice of Rh typing patients and blood donors by various serological methods. 17,32,36,[40][41][42] As the first step in a comprehensive review and evaluation of current options for updating policies for weak D testing and administration of Rh immune globulin in women with a weak D phenotype, the CAP TMRC conducted a survey in 2012, repeating the questions of the 1999 survey. The present article summarizes the results of the 2012 survey, compares them with those of the 1999 survey, and comments on the opportunities for updating policies and procedures based on changes that have occurred.…”