Introduction: Existence of hundreds of RHD gene variants contributes to variable D antigen expression and inconsistencies in reporting the RHD results. The aim of the study was to determine the serological and molecular characteristics of the most prevalent RHD alleles encoding serologically weak D variants. Material and Methods: Blood donors (n = 145 924) were typed for D antigen using the direct serologic micromethod. Nonreactive samples were analysed in IAT method with the IgM/IgG anti-D monoclonal blend, and 0,2% (n = 263) confirmed weak D antigen expression. After genomic DNA extraction (Qiaqen, Germany), RHD genotyping was performed using in house reagents and PCR-SSP kits (Inno-Train, Germany). Results: The prevalence of serologically weak D in blood donor population was 0.2% (n = 263). RHD genotyping confirmed weak D allele in 92.4% and partial D allele in 7.6%. The most common was weak D type 1 (49.7%) followed by weak D type 3 (24.7%) and type 2 (9.5%). Relatively high frequency was detected for weak D type 14 (4.6%) and type 64 (2.3%). In the category of partial D phenotypes, only DVI variant was found. Direct typing has shown great variability in the strength of reactions with different clones of anti-D reagents. Conclusion: Weak D type 1 is the most common weak D variant in Croatian blood donor population. The frequency of D variants and distribution of Rh phenotypes in our study was in concordance with other studies. It has been shown that serological methods and the combination of clones used, cannot distinguish variant D types, which justifies the use of molecular methods.

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IntroductionExposure 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.Material and MethodsBlood 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.ResultsAfter 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.ConclusionCurrently 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.