Demographics of Rhesus Phenotype of Blood Donors in Calabar: A Case Study of University of Calabar Teaching Hospital, Calabar, Cross River State, Nigeria

Background Rhesus antigens have been documented to cause haemolytic disease of the newborn as well as acute and delayed transfusion reactions. This study was performed to evaluate the frequency of rhesus antigens (C, c, D, E, and e) in the studied population. Method This study was a cross-sectional study involving 130 prospective blood donors attending University of Calabar Teaching Hospital (UCTH) donor clinic. Donors were grouped for Rh antisera (anti-E, anti-e, anti-C, anti-c, and anti-D) using the standard serologic technique. Result The most prevalent Rh antigen was “c” (98.5%), followed by “D” (97.7%), while the least was “C” (30.7%). The most prevalent phenotype was cDe/cDe (R0R0). Conclusion This work therefore concludes that the most prevalent rhesus antigen and rhesus phenotype was c and cDe/cDe among blood donors in University of Calabar Teaching Hospital.


Background
e term "blood group" refers to the entire blood group system comprising red blood cell antigens whose specialty is controlled by a series of genes which can be allelic or linked very closely on the same chromosome [1]. Presently, 33 blood group systems representing over 300 antigens are listed by International Society of Blood Transfusion [2,3]. e antigens may occur as integral proteins, where the polymorphism lies in the variation in amino acid sequence (e.g., rhesus (Rh) and Kell) or as glycoproteins or glycolipids (e.g., ABO) [1]. e phenotype of blood group of an individual is the observable expression of the genes inherited by the person and reflects the biologic activity of genes. e presence or absence of antigens on red cells is determined by serological testing representing the phenotype [4,5].
e rhesus blood group, formerly known as the rhesus system, is the second most important blood group system after ABO [6]. At a more comprehensive level, the rhesus system is considered as a gene complex that gives rise to various combinations of three alternative antigens C or c, D or d, and E or e, as originally suggested by Fisher [7]. e rhesus locus is located on chromosome 1 and comprised two highly homologous, very closely linked genes RhD, C, and E [8]. is concept of D, C, c, E, and e genes linked closely and transmitted together is consistent with Fisher nomenclature, and it is recommended by the World Health Organization Expert Committee in the interest of simplicity and uniformity [9]. e rhesus antigens are defined by corresponding antisera with the exception of "anti-d," which does not exist as it was thought to be amorphic without any corresponding antigen on red blood cells [8]. Anti-D is the most immunologically and clinically most important antibody in the rhesus system causing haemolytic transfusion and haemolytic disease in the newborn [10][11][12]. However, antibodies to the other Rh phenotype C, c, E, and e, to a lesser extent, cause haemolytic disease in the newborn and transfusion haemolytic reaction [10,13,14]. In routine transfusion practice in our study environment, Rh antigen typing is restricted to only D phenotype screening partly due to unavailability of antisera: C, c, E, and e and lack of policy to this effect [15]. Women of child bearing age and patients prone to recurrent blood transfusion in our environment and similar settings are at higher risk of developing haemolytic disease in the newborn and haemolytic transfusion reaction due to antibodies against these nonscreened Rh antigens, at times fatal [13,14]. Most of the studies done in the country (Nigeria) are restricted to the D phenotype. is study is aimed at filling this gap by providing the information on the prevalence of Rh phenotypes among blood donors as a baseline for policy formation and future planning towards safe blood transfusion and prevention of haemolytic disease of the newborn.

Methods
is study took a cross-sectional design with a systematic random sampling technique. A total of 130 prospective blood donors attending University of Calabar Teaching Hospital (UCTH) donor clinic were recruited into the study. Two millimeters (2 ml) of venous blood was collected from each of the 130 blood donors through veinpuncture using the antecubital vein into a plain container. e rhesus phenotypes were determined according to manufacturer's instructions using five specific monoclonal antisera (anti-D, anti-E, anti-C, anti-c, and anti-e) supplied by Lorne Laboratories (United Kingdom). e principle is based on the ability of Lorne reagents to cause a direct agglutination of the test red blood cells that carry the corresponding rhesus antigen. e presence of the group-specific rhesus antigen was indicated by agglutination.

Result
Of the five major antigens screened, "c" antigen was found to be the most common antigen (98 : 5%; n � 128), followed by the "D" antigen (97.7%; n � 127) and "e" antigen (95.4%; n � 124). e least observed antigen was "C" (30.7%; n � 40), whereas antigen "E" had a prevalence of 39.2% (n � 51) ( Table 1). e cDe/cDe phenotype had the highest distribution (46.2%; n � 60), followed by cDE/cde (20.6%; n � 26). e least observed phenotypes were Cde/cde, cde/cde, and CDE/ CDe with a prevalence of 0.8% (n � 1) ( Table 2). Table 3 shows the frequency of rhesus antigen observed in this study compared with published results in other parts of Nigeria and sub-Saharan Africa. Table 4 shows the comparison of the rhesus phenotype distribution in this study with other published data from studies in Nigeria.

Discussion
Data on various blood group antigens and phenotype frequencies in a population are essential in work-up plan for blood transfusion services [5].
We observed rhesus antigen "c" to be the most prevalent antigen with a prevalence of 98.5%. is finding is similar to the report of Jeremiah and Buseri and Jeremiah and Odumody [16,17] who reported the same antigen as most prevalent (99.8% and 100%, respectively) in studies done in Port Harcourt and Calabar, Nigeria. Antigens D (97.7%) and e (95.4) were the next common frequently occurring antigens, respectively. In contrast with the present study, both studies recorded a higher prevalence of antigen "e" than "D" [16,17]. However, the observation of this study is at variance with the report of Gwaram and Abdullah [15] who reported highest occurrence of antigen "D" in a study in Kano, Nigeria. Studies in other African countries such as Mauritania [18] reported antigen "e" (98.2) as the most prevalent rhesus antigen, while the study in Cote d'Ivoire [19] reported both antigens "c" (99.9%) and "e" (99.9%) as the most prevalent rhesus antigens. More so, studies outside Africa has reported antigen "e" (98.4%) in India [5], antigen "D" 99.0% in China, and antigen "e" (98%) in Black Americans as the most frequently occurring antigens. e disparity in the studies in Nigeria may be due to ethnic variations owing to heterogeneous nature of Nigeria. Nigeria has been described as a heterogeneous society with ethnic pluralism [20]. Rhesus antigens have been documented to vary among races [21]. Anti-D, anti-C, anti-E, and anti-c have all been implicated in haemolytic transfusion reactions, particularly delayed reactions [22]. Anti-D causes the most severe form of haemolytic disease of the newborn, and it is the major cause of fetal death. Another alloantibody capable of causing severe HDN includes anti-c [23,24].

Advances in Hematology
In our study, the most common phenotype was cDe/cDe (Dccee; R 0 R 0 ) followed by cDE/cde (DccEe; R 2 r). is finding is similar to previous studies in Port Harcourt and Calabar [16,17] that reported Dccee (cDe/cDe) as the most prevalent phenotype (60.8% and 73.61%, respectively). Other studies outside Nigeria have reported CDe/CDe (R 1 R 1 ), CDe/cDe (R 1 R 0 ), and cDe/cDe (R 0 R 1 ) as the most prevalent phenotypes in Asians [25,26], Caucasians [27], and Black Americans [25], respectively. However, cDE/cDE (R 2 R 2 ), CdE/cDe (r y R 0 ) cdE/cde (r 11 r), cdE/cdE (r 11 r 11 ), Cde/Cde (r 1 r 1 ), CdE/Cde (r y r 1 ), CdE/cdE (r y r 11 ), CdE/cde (r y r), and CdE/CdE (r y r y ) were not detected in the studied population. Due to the low sample size, we could not declare these phenotypes as rare blood. Rare blood is defined on the basis of blood group characteristics, as being found at a frequency of <1 : 1000 random samples in a given population [28,29]. e prevalence of RhD positive cases was 97.7% and n � 127, whereas the RhD negative was 2.3% (n � 3). This finding is similar to 97.1% and 93.9% reported in Kano [15] and Benin in Nigeria, respectively [30].

Conclusion
e prevalence of rhesus antigens and phenotypes showed similar pattern in this study and other previous studies with antigen c and cDe/cDe phenotypes occurring as most prevalent antigen and phenotype in Nigerians and other African population. e antigens were found in the order c > D > e > E > C. e data of these antigens put the call to incorporate their testing in synergy with the D antigen in routine blood screening prior to transfusion. We observed low frequency of rhesus D negative blood group in this study (2.3%). e low prevalence of RhD negative blood groups in our study environment also illuminates the need to make proactive plan in event of RhD negative patient requiring blood transfusion. Our frequency data on Rh antigens can help to implement different transfusions and obstetric strategies, which can ultimately improve our patient care. Mass scale typing, however, might be required to complete the database for Rh antigens in Nigeria.

Data Availability
Datasets generated and analyzed in this study are available from the corresponding author upon request.

Ethical Approval
is study was approved by Health Research Ethical Committee (HREC) of the University of Calabar Teaching Hospital.

Consent
Informed consent was obtained from the subjects.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
JEE conceived the study, sourced for literature materials, performed the laboratory analysis, and analyzed data. RAA supervised the data, performed laboratory analysis, and analyzed data. JOA supervised the workup and analyzed data. HUO did literature search, analyzed data, performed statistical analysis, and prepared the manuscript draft. All authors read and approved the final manuscript.