RHD variants in Polish blood donors routinely typed as D-

Abstract

Background: Blood donors exhibiting a weak D or DEL phenotypical expression may be mistyped D- by standard serology hence permitting incompatible transfusion to D- recipients. Molecular methods may overcome these technical limits. Our aim was to estimate the frequency of RHD alleles among the apparently D- Polish donor population and to characterize its molecular background.

Study design and methods: Plasma pools collected from 31,200 consecutive Polish donors typed as D- were tested by real-time polymerase chain reaction (PCR) for the presence of RHD-specific markers located in Intron 4 and Exons 7 and 10. RHD+ individuals were characterized by PCR or cDNA sequencing and serology.

Results: Plasma cross-pool strategy revealed 63 RHD+ donors harboring RHD*01N.03 (n = 17), RHD*15 (n = 12), RHD*11 (n = 7), RHD*DEL8 (n = 3), RHD*01W.2 (n = 3), RHD-CE(10) (n = 3), RHD*01W.3, RHD*01W.9, RHD*01N.05, RHD*01N.07, RHD*01N.23, and RHD(IVS1-29G>C) and two novel alleles, RHD*(767C>G) (n = 3) and RHD*(1029C>A). Among 47 cases available for serology, 27 were shown to express the D antigen

Conclusion: 1) Plasma cross-pool strategy is a reliable and cost-effective tool for RHD screening. 2) Only 0.2% of D- Polish donors carry some fragments of the RHD gene; all of them were C or E+. 3) Almost 60% of the detected RHD alleles may be potentially immunogenic when transfused to a D- recipient.

Figure 1

The flow of cross-pooling strategy done in case of RHD gene detection in the 48-pool prepared from plasma. Samples from each column or row of the archive plate are combined to create 14 cross-pools. After DNA isolation RHD detection of 14 samples reveals two RHDpos cross-pools (plasma from a column #D and a row #3). Crossing numbers of RHDpos column and RHDpos row allows to identified an individual RHDpos sample on the archive plate (in D3 position).

Figure 2

Nucleotide sequences for RHD(S256X) and RHD(Y343X). Pertinent nucleotide and amino acid sequences for: 767C>G in exon 5 (NG_007494.1, position:29144..29230) and 1029C>A in exon 7 (NG_007494.1, position:34176..34255).

Figure 3

An algorithm of classifying seemingly RhDneg donors to Dpos or Dneg group. *according to Rhesus base/ EMBL