RhD alloimmunization by DEL variant missed in donor testing

Exposure to normal or variably expressed RhD antigens in an antigen-negative individual can elicit an immune response and lead to the formation of clinically significant anti-D alloantibodies. We present the case of anti-D alloimmunization by DEL variant missed in routine blood donor screening.

Blood donors were typed for D antigen using the direct serologic micromethod. Nonreactive samples were confirmed in the indirect antiglobulin method with an IgM/IgG anti-D monoclonal reagent. Genomic DNA was extracted using a commercial QIAamp DNA Blood Mini kit on the QIAcube device (Qiaqen, Germany). RHD genotyping was performed using the PCR-SSP genotyping kits- Ready Gene D weak, Ready Gene D weak screen, Ready Gene CDE, and Ready Gene D AddOn (Inno-Train, Germany). Unidentified alleles were sent for DNA genome sequencing.

After identifying DEL positive blood units in RhD negative blood donor pool, a look-back study was performed to determine if their previous donations caused alloimmunization in recipients. Out of 40 D negative recipients, one developed anti-D alloantibody after 45 days. The patient did not receive other RhD positive blood products. Blood donor typed D negative in direct and indirect agglutination method. RHD screening was positive, but RHD genotyping and DNA sequencing showed no mutation indicating the normal genotype.

Currently used methods in RHD genotyping are insufficient to identify many variant alleles, especially intronic variations. We suggest additional gene investigation including yet unexplored regions of regulation and intron regions to justify our serological finding.