DNA analysis of blood samples with weak D antigen expression

LUJAN BRAJOVICH, MELINA; TRUCCO BOGGIONE, CAROLINA; RACCA, LILIANA; NOGUÉS, NÚRIA; MUÑIZ-DÍAZ, EDUARDO; COTORRUELO, CARLOS

Berlín

Congreso; XXXIst International Congress of the International Society of Blood Transfusion in joint cooperation with the 43rd Congress of the DGTI; 2010

International Society of Blood Transfusion

Background: Weak-D phenotypes express a significantly reduced amount of D antigen per red blood cell (RBC). These erythrocytes react with some anti-D reagents by direct agglutination techniques and often show a positive result by the indirect antiglobulin technique (IAT). Weak-D phenotypes emerge from RHD point mutations coding for transmembraneous or cytosolic RhD amino acid substitutions. It has recently been shown that carriers of weak-D types 1, 2 and 3 do not produce anti-D when exposed to D positive RBCs. The identification of patients with these phenotypes would avoid the wastage of D negative RBC units. Aims: The aim of this study was to characterize the molecular background of samples with weak D antigen expression in the population of Rosario, Argentine. Materials and Methods: We studied 59 blood samples that reacted with anti-D only by IAT. The Rh phenotype was performed by hemagglutination using specific monoclonal antibodies. DNA samples were initially screened using PCR SSP methods to detect weak D types 1, 2, 3 and 4 alleles. Variant samples not classified were further studied by RHD exon scanning, PCR RFLP and microarray strategies. We also screened weak D types 1, 2, 3 and 4 alleles in 56 samples that showed a weak agglutination by direct techniques. Results: From the 59 samples that reacted with anti-D only by IAT, 31 (52.55%) were weak D type 1; 14 (23.74%) weak D type 2; 4 (6.78%) weak D type 3 and 7 (11.86%) weak D type 4. In this group, we also identified 1 DVa, 1 DFR and 1 weak D type 59 by RHD exon scanning, PCR RFLP and microarray analyses. From the 56 samples with weak agglutination by direct techniques, 4 (7.14%) were weak D type 1; 3 (5.36%) weak D type 2; 4 (7.14%) weak D type 3 and 5 (8.93%) weak D type 4. These findings indicate that 94.92% (56/59) of the samples that reacted with anti-D by IAT and 28.57% (16/56) of the samples with weak direct agglutination belong to weak D types 1 to 4. Among these weak D samples, types 1, 2 and 3 represent 83.33% (60/72). The C antigen was present in 85.71% of weak D type 1 and in 87.50% of weak D type 3. The E antigen was observed in 76.47% of weak D type 2 and 91.67% of weak D type 4 were C and E negative. Conclusions: Molecular studies showed that the same weak D type may react either in saline or by IAT in different individuals and that the complete Rh phenotype can not be used to predict the weak D type, although we observed a strong correlation. PCR SSP strategies to detect weak D types 1 to 4 become appropriate for testing patients with weak expression of the D antigen in our population and would help to rationalize the use of D negative units for transfusion and anti-D prophylaxis in pregnant women.