Determining the Genotype of the RH Blood Group System in Maltese Blood Donors

Antonella Giordmaina Powney *

Hospital Blood Bank, Pathology Department, Mater Dei Hospital, Malta.

Joseph Borg

Department of Applied Biomedical Science, Faculty of Health Sciences, University of Malta, Malta.

Jesmond Debono

Hospital Blood Bank, Pathology Department, Mater Dei Hospital, Malta.

*Author to whom correspondence should be addressed.


Aims: The Rh blood group system is a complex protein based system having the D, C, c, E and e as the most immunogenic antigens. This blood group system has a vital role in transfusion medicine thus by DNA typing, important medical issues in transfusion practice can be resolved when serological techniques fail. The aim of the study was to determine the frequency of the RH genotype in the Maltese blood donors using molecular techniques.

Methodology: 797 blood donor samples were enrolled in this study. An allele-specific polymerase chain reaction (AS-PCR) method was used to determine the presence of RHD, RHCE*E and RHCE*e, while multiplex PCR was used to test for RHCE*C/c.  

Results: Out of 797 samples studied, the most common allele was RHCE*e with a percentage of 98.  The most common genotype in RhD positive samples was DCcee and in RhD negative samples, the most common was dccee. The most frequent haplotype was DCe.

Conclusion: Like in previous studies, this research also concludes that the distribution of the RH genotype varies in different geographical areas. Further studies working with larger sample size and better techniques would share better light on the molecular function of these loci. 

Keywords: Rh blood group system, RH frequency, blood group genotyping, allele specific PCR, multiplex PCR

How to Cite

Powney , A. G., Borg, J., & Debono , J. (2023). Determining the Genotype of the RH Blood Group System in Maltese Blood Donors. International Blood Research & Reviews, 14(4), 123–132.


Lasić L, Lojo-Kadrić N, Silajdžić E, Pojskić L, Hadžiselimović R, Pojskić N. Molecular – genetic variance of RH blood group system within human population of Bosnia and Herzegovina. Bosn J Basic Med Sci. 2013;1:10-13.

Cartron J. Defining the Rh blood group antigens. Biochemistry and molecular genetics. Blood Rev. 1994;8(4):199 -212.

Faas B, Simsek S, Bleeker P, MA O, Cuijpers H, Borne Av, Schoot Cv. Rh E/e genotyping by allele-specific primer amplification. Blood. 1995;829-832.

Castilho L, Pellegrino J. Blood group genotyping. Revista Brasiliera de Hematologia e Hemoterapia. 2004;26(2):135-140.

Cotorruelo C, Bondi C, Borras SG. Molecular Determination of RhD phenotype by DNA Typing: clinical applications. Ann Clin Biochem. 2000;37:781-789.

Flegel W. Molecular genetics and clinical applicationsn for RH. Transfus Apher Sci. 2011;1:81-91.

Govender L, Prakashchandra RD, Pillay P, Jentsch U. Molecular red cell genotyping of rare blood donors in South Africa to enhance rare donor-patient blood matching. African Journal of Laboratory Medicine. 2021;10(1).

Harry M, Deborah W. The gel test. Laboratory medicine. 1994;25(2):81-85.

Sandler SG, Flegel WA, Westhoff CM, Denomme GA, Delaney M, Keller MA, Simon CD. It's time to phase in RHD genotyping for patients with a serologic weak D phenotype. Transfusion. 2015;55(3):680-689.

Reesink HW, Engelfriet CP, Schennach H, Gassner C, Wendel S, Fontão-Wendel R, Yahalom V. Donors with a rare pheno (geno) type. Vox Sang. 2008;95:236-253.

Santiago R, Gaunt T, Day I. Hardy-Weinberg equilibrium testing of biological ascertainment for mendelian randomization studies. Americal Journal of Epidemiology. 2009;505-514.

Cohen J. A coefficient of Agreement for Nominal Scales. Educaitional and Physiological Measurment. 1960;20(1):37-46.

Barret J, Fry B, J, M, Daly M. Haploview: analysis and visualization of LD and haplotype maps. Bionformatics. 2005;21(2):263-265.

Simsek S, Faa B, Bleeker P, Overbeeke M, Cuijpers H, Schoot CV, Borne AV. Rapid RHD genotyping by polymerase chain reaction based amplification DNA. Blood. 1995;85(10):2975-2980.

Hojjati M, Einollahi N, Nabatchian F, Pourfathollah AA, Mahdavi MR. Allele-specific oligonucleotide polymerase chain reaction for the determination of Rh C/c and Rh E/e antigens in thalassaemic patients. Blood Transfusion. 2011;9:301-305.

Krishna GD, Sreedhar VK, Arun R, Jothibai SD. Prevalence of principal Rh Blood Group antigens in blood donors at the blood bank of a tertiary care hospital in Southern India. Journal of Clinical and Diagnostic Research. 2016;10(5).

Lin-Chu M, Broadberry R, Chang F. The distribution of blood group antigens and alloantibodies among Chinese in Taiwan. The Journal of AABB Transfusion. 1988;28(4):350-352.

Yu Y, Ma C, Sun X, Guan X, Zhang X, Saldanha J, Wang D. Frequencies of red blood cell major blood group antigens and phenotypes in the Chinese Han population from Mainland China. International Journal of Immunogenetics. 2016;226-235.

Vuhahula EA, Yahaya J, Morgan ED, Othieno E, Mollel E, Mremi A. Frequency and distribution of ABO and Rh blood group systems among blood donors at the Northern Zone Blood Transfusion Center in Kilimanjaro, Tanzania: a retrospective cross-sectional study. British Medical Journal Open; 2023.

Doku G, Agbozo W, Annor R, Mawudzro P, Agbeli E. Frequencies and ethnic distribution of ABO and RhD blood groups in the Volta region of Ghana, towards effective blood bank services. African Health Science; 2022.

Daniels G. RH and RHAG blood group systems. In D. G, Human Blood Groups UK: Blackwell Publishing. 2013;182-258.

Wagner F, Kasulke D, Kerowgan M, Flegel W. Frequencies of the blood groups ABO, Rhesus, D category VI, Kell and of clinically relevant high frequency antigens in South- Western Germany. Infusionsther Transfusionsmed. 1995;22 (5):285- 290.

Weinstock C. It is worthwhile filling in the remaining blank spots for blood group antigen frequencies. Blood Transfusion. 2014;12(1):3-6.

Reid M, Lomas-Francis C, Olsson M. The Blood Group Antigen Facts Book. (3rd, Ed.) Academic Press; 2012.

Gundrajukuppam DK, Vijaya SB, Rajendran A, Sarella JD. Prevalence of principal Rh blood group antigens in blood donors at the blood bank of a tertiary care hospital in Southern India. Journal of Clinical and Diagnostic Research. 2016;10 (5):7-10.

Kahar MA, Patel RD. Phenotype frequencies of blood group systems (Rh, Kell, Kidd, Duffy, MNS, P, Lewis, and Lutheran) in blood donors of south Gujarat, India. Asian Journal of Transfusion Science. 2014;8(1):51-55.

Makroo R, Gupta R, Bhatia A, Rosamma NL. Rh phenotype, allele and haplotype frequencies among 51,857 blood donors in North India. Blood Tranfsusion. 2014; 12(1).

Sarkar R, Brig Philip J, Col b, Mallhi R, Cdr S, Comdt D. Proportion of Rh phenotypes in voluntary blood donors. Med J Armed Forces India. 2013;69(4):330-334.

Arwa AR, Ali AM, Saif AH, Sabah AM, Michael S, Sheila O, Murtadha AK. Prevalence of red blood cell major blood group antigens and phenotypes among omani blood donors. Oman Medical Journal. 2019;34(6):496-503.

Hassan HA, Maymoon M, Gasim D, Aymen MM, Basem M, Yahia H, Muhammad S. Prevalence of Rh and K phenotypes among blood donors from different ethnicities in Samtah (Southern Region) Saudi Arabia. International Journal of Immunogenetics; 2022.

Taha JY. Rh Antigen and phenotype frequency in Kalba Region, UAE. Bahrain Medical Bulletin. 2012;34(1).

Bogui LS, Dembele B, Sekongo Y, Abisse Y, Konate' S, Sombo M. Phenotypic Profile of Rh and Kell Blood Group Systems among Blood Donors in Cote d'Ivoire, West Africa. Journal of Blood Transfusion; 2014.

Jeremiah ZA, Buseri FI. Rh antigen and phenotype frequencies and probable genotypes for the four main ethnic groups in Port Harcourt, Nigeria. Immunohaematology. 2003;3:86-88.

Costa DC, Schinaider AA, Santos TM, Schörner EJ, Simon D, Maluf SW, Silva MC. Frequencies of polymorphisms of the Rh, Kell, Kidd, Duffy and Diego systems of Santa Catarina, Southern Brazil. Rev Bras Hematol Hemoter. 2016;199-205.

Farjal T, Javeria A, Naveena AR, Yumna A, Munira B. The frequency of Rh phenotype and its probable genotype. Cureus. 2022;14(6).

Baruah D, Devi G, Musfique J, Bharali A, Dutta UC. Distribution and frequency of principal Rh blood group antigens (D, C, c, E, and e) and their phenotypes in the blood donors attending blood bank in a tertiary care hospital in Barpeta district of Assam. sian Journal of Transfusion Science. 2022;16(2).

Race R, Mourant A, Lawler SD, Sanger R. The Rh chromosome frequencies in England. Blood. 1948;3(6):689-695.

Mohrah A, Yasser A, Rana H, Abdulkareem A, Ammar A, Abdulmajeed A, Omar A. Frequency of Rh and K antigens in blood donors in Riyadh. Hematology, Transfusion and Cell Therapy. 2022;44 (4):555-559.

Ben HA, Raoudha B, Pedro M, Esther E, Hassen C. Variations of Rhesus Haplotype Frequencies in North Africans and in Worldwide population Analyses. International Journal of Human Genetics; 2015.

Belsito A, Costa D, Fiorito C, Iorio GD, Casamassimi A, Perrotta S, Napoli C. Erythrocyte genotyping for transfusion-dependent patients at the Azienda Universitaria Policlinico of Naples. Transfusion and Apherisis Science. 2015;52(1).

Chou S, Evans P, Vege S, Coleman S, Friedman D, Keller M, Westhoff C. RH genotype matching for transfusion support in sickle cell disease. Blood, The Journal of the American Society of Hematology; 2018.

Handa A, Kukar N, Maharishi R, Syal N, Arora H. Analysis of red cell alloimmunization in multi transfused patients at a Tertiary care teaching hospital. Journal of Family Medicine and Primary Care; 2020.

Floch A. Molecular genetics of the Rh blood group system: Alleles and antibodies—a narrative review. Annals of Blood; 2021.

Yang S, Liqin W, Jiameng N, Ting M, Lili X, Aowei S, Jiangcun Y. Distribution characteristics of ABO blood groups in China. Heliyon; 2022.