RHD Allelic Identification Among D−Brazilian Blood Donors as a Routine Test Using Pools of DNA

Abstract

Background RHD alleles leading to a reduced expression of D antigen of the red blood cell (RBC) surface may be erroneously typed as D− by serology and may cause anti‐D immunizations when transfused to recipients. Methods To determine the occurrence of such alleles among apparent D− blood donors, molecular typing was implemented as a routine test using a pool of DNA. A total of 2,450 pretyped D− samples were tested in pools of 10 for the RHD‐specific polymorphism in intron 4 and exon 7. Samples in polymer chain reaction (PCR) positive pools were individually reevaluated by exon‐specific PCRs, sequencing, and serologic methods. Results Among 2,450 serologically D− blood donor samples tested, 101 (4.1%) carried the RHD gene. Nonfunctional RHD (RHDψ, RHD*CE(2–9)‐D, and RHD*CE(3–7)‐D), different weak D alleles such as RHD*weak D type 1, RHD*weak D type 4.3, RHD*weak D type 5, RHD*weak D type 38, and RHD*DEL were identified. Conclusion We employed a PCR‐based assay for RHD as a routine test using pools of ten DNA blood donor samples. The integration of RHD genotyping into the routine screening program using pools of DNA samples was straightforward. As a consequence, 19 (0.8%) blood donors carrying a weak D and Del phenotypes with the potential of causing anti‐D immunizations in recipients were reclassified as D+. For each population, it would be necessary to adapt the RHD genotyping strategy to the spectrum of prevalent alleles.

Evaluation of the applicability and effectiveness of a molecular strategy for identifying weak D and DEL phenotype among D– blood donors of mixed origin exhibiting high frequency of RHD*Ψ

“…In this sample, the most frequent DEL allele was RHD*DEL1 , which was identified in 50% of donors with this phenotype. This allele is very common among East Asians, comprising almost one‐third of their D– population, and is designated as c.1227G>A or Asia DEL in the medical literature . In our cohort, only one of the donors exhibiting RHD*DEL1 allele self‐declared as Asian.…”

“…To avoid RhD sensitization, D– donors should be checked by RHD polymerase chain reaction (PCR) aiming to exclude weak D and DEL donors who may have been lost by serology. Diverse RHD genotyping protocols have been successfully applied worldwide, most aimed at the amplification of one or more gene regions ( RHD promoter, RHD Intron 4, RHD Exon 7, and RHD Exon 10) of phenotypically D– donors, either in single samples or in sample pools . As the frequency of the DEL phenotype is higher among C+ and/or E+ donors (1:67) in comparison to C– and E– donors (1:43,053), excluding the latter from the screening protocol represents a cost‐effective choice .…”

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

“…RHD genotyping can help to identify variants and to predict RhD phenotype. Molecular RHD strategies target RHD‐specific polymorphism . If a novel inactive RHD allele is encountered, prediction from genotype can lead to false‐positive phenotype.…”

<b><i>RHD</i></b> PCR of D-Negative Blood Donors

“…Another difference to conventional RHD PCR applications is the possibility to start with pooled testing [8,28,30,35,39,48]: As long as the samples have initially be checked at least by direct agglutination with anti-D, in Caucasian populations only a minority of samples will be RHD -positive.…”