Naslov
Molekularno određivanje alela RHD u populaciji serološki RhD negativnih davatelja krvi sjeverozapadne Hrvatske
(Molecular identification of RHD alleles in serologically RhD negative blood donors from northwestern Croatia)

Autori
Lilić, Marko

Vrsta, podvrsta i kategorija rada
Ocjenski radovi, doktorska disertacija

Fakultet
Odjel za biologiju

Mjesto
Osijek

Datum
09.05

Godina
2022

Stranica
153

Mentor
Golubić Ćepulić, Branka ; Samardžija, Marina

Ključne riječi
varijante gena RHD, antigeni sustava Rh, serološki RhD negativni darivatelji krvi
(RHD gene variants, Rh system antigens, serological RhD negative blood donors)

Sažetak
D antigen is highly immunogenic, the highest after A and B antigens. It can induce anti D antibody production if it is recognized as foreign by the immune system of an RhD negative person due to transfusion, pregnancy or transplantation. Some D variants, especially those expressed by hybrid alleles, can react falsely negative in routine serology tests for D antigen determination. False reactivity could be caused by the lower number of D antigens on erythrocyte membrane or not enough D epitopes within the products of hybrid Rh alleles. Irrespective of the cause of the negative serological reaction, D variants can immunize RhD negative individuals. These clinical reasons and accelerating development of molecular diagnostics in the last decade, most notably for Rh alleles, prompted an increasing number of institutes for transfusion medicine to implement a method of molecular screening for RHD in RhD negative blood donors. Aims of this study were to report and describe RHD variants in RhD negative blood donors of northwestern Croatia, their frequencies and haplotypes with RHCE, interpret the discordant serology and molecular results as well as to propose the algorithm for routine Rh alleles testing implementation. In this two-year prospective study, the participants were 704 unrelated voluntary blood donors from Međimurje and Varaždin County in northwestern part of Croatia. All blood donors were serologically RhD negative and were individually tested for RHD exons. RHD screening was performed by examination of RHD exons 3, 5 and 10 in 589/704 (83.7%) serologically RhD negative ccee samples. RHD deletion was established in all of them. Serology results of the Rh phenotype (C+ and/or E+) were confirmed by qPCR in the remaining 115 participants (16.3%). In this group, 112 donors were confirmed as RHD negative as well, because none of the RHD exons were reactive. RHD DEL alleles were determined in two Ccee donors (0.28%): RHD*DEL32 (with single nucleotide polymorphism in intron 1, c.149-29G>C) and RHD*DEL44 (of structure RHD*D-CE(4-9)-D). One participant (0.14%) was proven as homozygous for RHD deletion, but simultaneously revealed one distinct RHD sequence in 5'UTR within RHCE locus, c. 132A. It was detected in a hybrid allele with genomic structure RHCE*D-CE(2-10), a subtype of RHCE*02 allele. All three donors with Rh variants expressed D antigens or RhD epitopes as displayed by the adsorption and elution test. The frequency of each Rh allele in RhD negative blood donors is 0.07% or 1:1408 (95% confidence interval 1:55608 – 1:253). The RHD DEL alleles were proven for the first time in this study in a Croatian population. According to the published literature, this study was the first outside China to report a DEL phenotype of the antigen expressed by the allele RHD*DEL44, and particularly in a population of European origin. Hybrid allele RHCE*D-CE(2-10) has been described previously in China and Slovenia. This study demonstrated for the first time the presence of an RhD epitope or epitopes in a donor with homozygous RHD deletion containing this hybrid allele. Considering the proximity with the reported cases of the allele from the neighbouring Slovenia, it is plausible, that there could be a local basin of this hybrid allele. As the new NGS methods have been penetrating the field of immunohematology recently, with SMRT sequencing especially seeming encouraging for the near future, they could connect the complete genomic structure of the allele with the verified RhD epitope(s) in this particular sample from this study. The study plan and its results laid the foundation of the algorithm for implementation of Rh molecular testing within the blood donors in Croatia. Molecular Rh testing of the blood donors will provide more safety for patients who receive their blood products. Additionally, this would prevent the immunization of genuine RhD negative patients by an RhD positive unit, which reacted negatively in serological testing.

Izvorni jezik
Hrvatski

Znanstvena područja
Biologija, Temeljne medicinske znanosti, Kliničke medicinske znanosti, Interdisciplinarne biotehničke znanosti, Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)

POVEZANOST RADA