First Report of Phenotype Prevalence of Five Major Rh Blood Group Antigens in the Blood Donor Population of Peshawar, Pakistan

Objective: The current study investigated the prevalence of five major Rh blood group antigens in the Peshawar region of Khyber Pakhtunkhwa province, Pakistan.
Methodology:This was a cross-sectional study, conducted at the Peshawar Regional Blood Centre from February to April 2023. A total of 13,148 blood donor samples were analyzed for Rh D, C, c, E, and e antigens using monoclonal reagents. The frequencies were calculated by direct counting on a spreadsheet programme and the Statistical Package for Social Sciences was used to conduct a meta-analysis for comparison with national and international studies.
Results:The findings of the current study revealed that 92.17% of donors were RhD positive. The e antigen showed the highest frequency of 98.31%, followed by D antigen and the C antigen (92.17% and 78.37%, respectively). Comparative analyses with earlier Pakistani studies and international data indicated significant variations in antigen frequencies, potentially attributed to the diverse ethnic population and geography of the region.
Conclusion:The findings emphasize the importance of local antigen frequency data for efficient blood transfusion strategies, especially for thalassaemia patients requiring frequent transfusions.
Key words:Rhesus, Blood Group, Phenotype

The phrase "blood group" often describes the unique combination of surface antigens on red blood cells (RBCs) in an individual. Blood group antigens on red blood cells (RBCs) are hereditary, polymorphic structures made of proteins or carbohydrates that are found on the extracellular layer of the RBC membrane. They contribute to the architecture of the RBC membrane, and their specific function(s) include: membrane transporters (Diego, Kidd), receptor and adhesion molecules (Duffy, Lutheran), complement regulatory glycoproteins (Cromer, Knops), enzymes (Yt, Kell, Dombrock), structural components (Diego, Gerbich) or components of the glycocalyx (MNS).1,2 Our knowledge of blood groups has expanded over time to include specific disease associations with RBC surface antigens as well as transfusion-related issues.3-6
Technically, blood group systems are described as complexes of two or more closely related homologous genes or systems of one or more antigens controlled by a single gene. Every blood group system is genetically distinct from the others. To identify a blood group system and its antigens, the underlying genetic variation that influences phenotype needs to be found, sequenced, and verified.^7,8 The International Society of Blood Transfusion (ISBT) has identified 45 blood group systems containing 360 red cell antigens (a.o. July 2023).⁹ Rh is one of the most clinically significant and complex blood group systems, second only to the ABO blood group system. It’s warm-reacting antibodies can result in mild to severe haemolytic transfusion reactions as well as haemolytic diseases in foetus and newborn.10 The frequency and distribution of Rh antigens vary greatly across the globe. Less research has been done in Pakistan on the frequencies and immunogenicity of C, c, E, and e. Besides, there are no reports available from the Khyber Pakhtunkhwa province regarding the distribution of the major Rh blood group phenotypes.
Pakistan is the fifth most populated country in the world, with a wide range of ethnic backgrounds, especially in the province of Khyber Pakhtunkhwa, which has a long history of immigrants due to its proximity to the Afghan border. Due to consanguineous marriages11 and the lack of a national policy on thalassaemia prevention,12 the province of Khyber Pakhtunkhwa has a higher frequency of thalassaemia than other parts of the country.¹³
Consequently, the mainstay of treatment for people with thalassaemia major is multiple transfusions. In these patients, alloimmunization occurs frequently and the likelihood of alloimmunization rises with the frequency of transfusions.¹⁴ The majority of blood banks in Pakistan currently solely use random cross-matching of available inventory units to provide patients with appropriate blood. Therefore, in the absence of a local donor database, providing packed red blood cells that are phenotypically matched could be difficult. In order to identify antigen-negative blood in blood centres, data for the major Rh blood group phenotypes in various ethnicities have been thoroughly examined and utilized as a reference. There have only been five studies in Pakistan to date that have assessed the frequency of Rh major antigens; however, no data from the north-western part of the country has ever been reported. This encouraged us to carry out this study in order to determine the prevalence of Rh D, C, c, E, and e antigen frequencies in the Peshawar region of Khyber Pakhtunkhwa province, the north-western part of Pakistan. The objective is to enable the provision of antigen-negative blood to patients with alloantibodies and will help to make cost-effective use of blood centre resources and shorten turnaround times for phenotyping and crossmatching.

The Regional Blood Centre Peshawar, attached to six tertiary teaching hospitals, performed this prospective and cross-sectional study from February to April 2023. Ethical approval was granted by the Institutional Review Board of Regional Blood Centre Peshawar. The sample size was calculated by using WHO sample size calculator by using prevalence of principal Rh blood group antigen - E 18.8%, confidence level 95%, and absolute precision of 3% and sample size turned out to be 652. However, additional testing of samples was performed to include all blood donors during the study period. Hence, a total of 13,148 blood samples were randomly collected from healthy male and female blood donors for Rh antigen typing. A 5% red cell suspension was prepared in 0.9% normal saline.
The Rh D, C, c, E, and e antigens phenotyping was performed by standard tube technique using monoclonal reagents (Bio-Rad Laboratories) and tested against known positive (heterozygous antigens from antibody screening cells) and negative antigens according to manufacturer instructions. The frequencies were calculated by direct counting on a spreadsheet programme and the Statistical Package for Social Sciences (SPSS for Windows version 25.0. Armonk, NY, USA) was used to conduct a meta-analysis for comparison of results with national and international studies. All results were standardized to percentages.

A total of 13,148 blood donor samples were typed for five major antigens of Rh blood group system including D, C, E, c, and e. Of these, 92.17% of the donors were RhD positive and 7.83% were RhD negative. Table 1 shows the antigen frequency of the Rh antigens in our study. The e antigen showed the highest frequency of 98.31%, followed by D antigen and C antigen (92.17% and 78.37%, respectively). The results were compared with five earlier studies conducted in Pakistan and those conducted in neighbouring countries (Table 2 & 3).

The results of current study indicated a significant difference in the frequency of antigens E, C, and c between our population and those reported from Pakistan15-19 (Table 2).
This may be attributed to diverse ethnic population and geography of our region. It is pertinent to mention that the combined sample size of these five studies was 1,694, much less than our sample size of 13,148. When compared with studies from other countries, our findings indicated a statistically significant difference in the frequency of D, C, c, and E antigens.
In the Türkiye25 population, the D antigen frequency was 75% much less than our study (92.16%). The C antigen frequency showed a wide variation in the distribution; 59% in Saudi Arabia24 to 91.0% in India22 while our study reported a frequency of 78.36%. Antigen c showed the highest frequency (86%) in Saudi Arabian24 population whereas lowest frequency was seen in our study 45.69%. Likewise, the E antigen’s frequency varied between 14% in Türkiye25 to 50.78% in China20 (Table 3).
The data from current study may be used to help blood centres prepare in-house antibody identification extended panels to detect unexpected multiple antibodies when commercial antibody identification kits are not conclusive. The comparison of the study findings demonstrated that various regions of Pakistan have different frequencies of Rh blood group phenotypes. Given the high prevalence of the thalassaemia in the Peshawar region of Khyber Pakhtunkhwa province, the challenge for hospital blood banks and thalassaemia centres is to supply antigen-negative blood units to thalassaemia patients who need frequent transfusions as part of their treatment plan. In order to prevent alloimmunization, it is important to ascertain the antigen frequencies of the local donor population.²⁶

Utilizing a multiethnic cohort, this study represents a pioneering effort in delineating the prevalence of the primary Rh antigens within the specific geographic context of Pakistan. It stands as the inaugural investigation to provide comprehensive insights into the distribution of major Rh antigens in Peshawar region, contributing valuable data to the
existing body of knowledge. This research not only addresses a critical gap in understanding the immunogenetic landscape of Pakistan but also sets a precedent for future studies aiming to explore the diverse genetic variations within multiethnic populations in this part of the world.

1. Hosoi E. Biological and clinical aspects of ABO blood group system. J Med Invest. 2008;55(3-4):174-82. https://doi.org/10.2152/jmi.55.174. Daniels G. Functions of red cell surface proteins. Vox Sang. 2007;93(4):331-40. https://doi.org/10.1111/j.1423-0410.2007.00970.x. Zhong X, Wang DL, Xiao LH, Mo LF, Luo XF. ABO blood groups and nosocomial infection. Epidemiol Infect. 2023;151:e64.
2. Cano EA, Esguerra MA, Batausa AM, Baluyut JR, Cadiz R, Docto HF, Encabo JR, Gomez RM, Sadang MG. Association between ABO Blood Groups and Type 2 Diabetes Mellitus: A Meta-Analysis. Curr Diabetes Rev. 2023;19(6):e270422204139. https://doi.org/10.2174/1573399818666220427124448.
3. Davison GM, Hendrickse HL, Matsha TE. Do Blood Group Antigens and the Red Cell Membrane Influence Human Immunodeficiency Virus Infection? Cells. 2020;9(4):845. doi: 10.3390/cells9040845.
4. Gorakshakar A, Gogri H, Ghosh K. Evolution of technology for molecular genotyping in blood group systems. Indian J Med Res. 2017 Sep;146(3):305-315. https://doi.org/10.4103/ijmr.IJMR_914_16.
5. Blood Group Systems. International Society of Blood Transfusion (ISBT) Working Party for Red Cell Immunogenetics and Blood Group Terminology (ISBT WP). Available at https://www.isbtweb.org/isbt-working-parties/rcibgt.html accessed on Nov 20, 2023
6. Sarwar N, Nawaz R, Abid S, Hamza A, Khan A, Durrani A, Khan WS. Prevalence of congenital diseases among consanguineous marriages in District Peshawar Pakistan. Annals of Allied Health Sciences 2022;8(1):28-31. Zaheer HA, Waheed U. Development of a National Thalassemia Policy in Pakistan. Global Journal of Transfusion Medicine 2017;2(1):69-70. https://doi.org/10.4103/GJTM.GJTM_53_16
7. Karim F, Moiz B, Muhammad FJ, Ausat F, Khurshid M. Rhesus and Kell Phenotyping of Voluntary Blood Donors: Foundation of a Donor Data Bank. J Coll Physicians Surg Pak. 2015;25(10):757760-760. https://doi.org/10.2015/JCPSP.757760
8. Shah A, Jariwala K, Gupte S, Sharma P, Mishra K, Ghosh K. Pattern of distribution of 35 red cell antigens in regular voluntary blood donors of South Gujarat, India. Transfus Apher Sci. 2018;57(5):672-675. https://doi.org/10.1016/j.transci.2018.08.005.
9. Owaidah AY, Naffaa NM, Alumran A, Alzahrani F. Phenotype Frequencies of Major Blood Group Systems (Rh, Kell, Kidd, Duffy, MNS, P, Lewis, and Lutheran) Among Blood Donors in the Eastern Region of Saudi Arabia. J Blood Med. 2020;11:59-65. https://doi.org/10.2147/JBM.S236834.
10. tsadze I, AKhvlediani L, Khukhunaishvili R, Koridze M, Tskvitinidze S, NakaShidze I, et al. Specific Combinations of Erythrocyte Group Antigens in Blood Donors. Indian J Hematol Blood Transfus. 2020;36(2):356-360. https://doi.org/10.1007/s12288-019-01197-4