Supervisors info:
Πολίτου Μαριάννα, Αναπληρώτρια Καθηγήτρια, Ιατρικής Σχολής , Ε.Κ.Π.Α.
Βαλσάμη Σερένα, Επίκουρη Καθηγήτρια,Ιατρικής Σχολής, Ε.Κ.Π.Α.
Γιαλεράκη Αργυρή, Αναπληρώτρια Καθηγήτρια ,Ιατρικής Σχολής Ε.Κ.Π.Α.
Summary:
Rh blood group system is the most polymorphic and immunogenic blood group system, and along with the ABO the most significant for clinical practice. The RhD antigen, a complex mosaic of different epitopes, is the most important in the Rh system and is determined in all the blood donors and patients. Although most people are either RhD positive or RhD negative, there is a certain number of people who have a variation of the D antigen, which are called weak D, partial D and DEL phenotypes. Blood donor typing should account for all expressing antigens in order to prevent anti-D alloimmunization. Therefore, highly reliable routine D typing methods must be performed, taking into account the RhD discrepancies caused by variable reactivity of weak D and partial D types with different commercial antisera, as well as the very low antigen density of some of the RhD variants.
Study design: The objective of this prospective study was to determine the relative distribution of D variants among blood donors and patients coming to attention by weak agglutination in RhD with routine serology in a Greek tertiary hospital. Furthermore, the study aimed to investigate RHD alleles among blood donors who typed D- by serologic typing and positive for C and/or E. For this reason a previously published multiplex polymerase chain reaction (PCR) procedure was modified and used for the detection of RHD gene. Positive samples were further characterized by two commercial PCR-SSP kits.
Results: Of 27251 individual blood donors and 36551 patients within a 30 month period, 140 samples with atypical D antigen expression were further RHD genotyped by PCR-SSP technique. Among these, 56 samples (40%) were identified as weak D type 1, 18 (12.86%) as weak D type 3, 2 (1.43%) as weak D type 4, 12 (8,57%) as weak D type 5. An additional 16 samples (11.43%) were identified as partial D, 17 samples (12.14%) represented the DEL[M295I] genotype and 1 sample (0.71%) showed the RHD-CE(4-7)-D hybrid allele. The remaining 16 samples (11.43%) could not be discriminated with the available kits. In the second part of the study genomic DNA from 112 blood donors serologically typed as D- but C+ and/or E+ were tested individually for the presence of RHD-specific DNA sequences in the RHD promoter, intron 4, exon 7 and exon 10. Molecular analysis showed that 104 (92.86%) were negative for all four RHD DNA regions. Among the other 8 samples (7.24%), all of Ccee phenotype, three were found to be positive for RHD promoter, intron 4, exon 7 and exon 10, three for RHD promoter and exon 10, and two for exon 10 alone. Further genotyping revealed five hybrid RHD-CE-D alleles [3 RHD-CE(2-9)-D and 2 RHD-CE(3-7)-D], one allele represented the DEL(M295I) genotype, while the remaining two samples gave inconclusive RHD genotyping results using the PCR-SSP tests and need further investigation by sequencing.
Conclusions: The results from the molecular testing on frequency of D variants in the population of our hospital is in accordance with the results in the populations of other European countries, regarding weak D 1 and 3, though they show a significant percentage of weak D type 5 and DEL(M295I). Serotyping is the standard method to assign transfusion strategies but it is not always capable to correctly define all samples with D variants or reduced amount of D antigen. In order to efficiently support routine serologic D typing, a RHD genotyping strategy is needed to identify D variants with potential for anti-D alloimmunization and to confirm D- blood donors with clinically relevant alleles. Patients belonged to certain molecular categories can be treated as RhD-positive and thus valuable RhD-negative blood reserves can be preserved.
Keywords:
Rhesus D, Weak D, partial D, PCR, Blood Donors
