Prevalence of Rhesus Negative Status Amongst Antenatal Atendees in the University of Uyo Teaching Hospital, Uyo, Nigeria: A 5-Year Review

Introduction: The Rhesus (Rh) antigen is the most polymorphic of human blood group systems second only to the ABO in transfusion and transplantation medicine with significance in iso-immunization which can cause haemolytic disease of the foetus and newborn. This study was carried out to determine the prevalence of Rhesus negativity amongst antenatal clinic attendees in the University of Uyo Teaching Hospital. Methodology: This was a 5-year retrospective study carried out between March 1, 2012 and February 28, 2017. Data containing the ABO and Rhesus blood group of all antenatal clinic attendees was collected from the Laboratory registers and analysed using Microsoft excel. Results: The prevalence rate of Rhesus D negative women was 4.40%. The average yearly attendance was 2861 women. The commonest blood group was O with 8232 (57.54%), followed by blood group A, 3416 (23.88%) and blood group B, 2388 (16.69%) while the least common blood group was AB, 271 (1.89%). Most Rhesus D-negative women were of blood group O; 382 (60.78%), followed by group A; 142 (22.58%), and blood group B was 94 (14.49%). AB blood group was the least; 11 (1.70%). Sensitization rate among the 629 Rhesus D-negative women was 1.11%. Conclusion: The prevalence of rhesus negativity is low in the population studied with an ABO distribution that mirrored the antenatal population, hence the overall need for immunoprophylaxis. It is however very necessary to educate and counsel all non-sensitized Rhesus negative women on the need for antenatal and post-partum immuno-prophylaxis as well as public enlightenment on the rationale for Rhesus typing, especially among women.


Introduction
The Rhesus (Rh) blood group system is the most polymorphic of the human blood groups, believed to consist of at least 45 independent antigens and, is next only to the ABO in clinical significance and transfusion medicine. [1] It is a product of Rh antigens (include D, C or c, and E or e) which are trans-membrane proteins expressed on the surface of erythrocytes apparently used for the transport of CO 2 and/or ammonia across the plasma membrane. [2] Historically, following the description of the A, B, O blood types, a fourth, the Rhesus system was discovered by Landsteiner and Weiner in 1940. [3][4][5] This followed experiments which made use of Rhesus monkeys, hence the statuses of Rhesus Positive or Rhesus negative depending on the presence or absence of the antigen respectively on the red blood cells of a person. [1,3,4] The significance of the Rh status in pregnancy is the Rh D iso-immunization which can occur when a Rh D negative woman is pregnant with a Rh D Positive foetus and in-utero interaction between maternal and foetal blood from fetomaternal haemorrhage during the course of pregnancy. [6,7] This may arise either from spontaneous or induced abortion, trauma, normal delivery and invasive obstetric procedure or when an Rh-negative female receives an Rh-positive blood transfusion. [8,9] Anti-Rh D Immunoglobulin antibodies which are generated as a result of the iso-immunisation can cross the placenta once present and attack the foetal red blood cells resulting in a range of complications termed Haemolytic Disease of the foetus and newborn which could range from hyperbilirubinaemia through severe anaemia to Hydrops fetalis. [7][8][9] The maternal Rh immunoglobulin G (IgG) antibodies produced following iso-immunisation persist for life and may undergo transplacental migration to the foetal circulation with resultant formation of antigenantibody complexes with Rh-positive foetal red blood cells and subsequently resulting in the foetal alloimmune-induced haemolytic anaemia described. [8,10,11] Other causes of fetomaternal haemorrhage include amniocentesis, spontaneous or induced abortion, cordocentesis, chorionic villus sampling, and ruptured ectopic pregnancy. [1] The D-antigen is the most immunogenic and most commonly involved antigen in Rhesus incompatibility and Rh Haemolytic disease of newborn (HDN) despite the existence of different Rh blood group antigen subtypes (D, C, c, E, e). [8] The ABO group incompatibility is however the commonest cause of HDN, followed by anti-Rh D, while anti-Kell is the least involved. [10,12] Haemolytic disease of foetus and newborn following allo-immunization to Rh D is a major contributor to perinatal mortality and morbidity, hence impacting on a women's obstetric career. Sensitisation, anti D antibody formation and the sequale of Haemolytic disease of foetus and newborn can be prevented by the administration of anti-D immunoglobulin G (IgG) prophylaxis soon after delivery in Rhesus negative women and is recommended by the World Health organization (WHO). [11,13,14] Despite documented benefits, [9-11, 13, 14] uptake of anti-D immunoglobulins in Uyo, Nigeria is poor, largely due to prohibitive cost with a dose of Rhogam (Kedrion Biopharma, New Jersey, USA) costing N38,000 ($124). A search for National data on the prevalence of Rh D Negative women and the incidence of allo-immunization yielded no results with only few single centre studies. This study was carried out to with the aim of determining the prevalence of Rh D Negative blood group among antenatal clinic attendees in the University of Uyo Teaching Hospital located in South south Nigeria. This we hope will not only add to the data on this study subset, but will assist management in planning for, sourcing and making available the needed Rh immunoglobulin-G for prevention of alloimmunisation with a view to eliminating Haemolytic disease in Fetuses and Erythroblastosis fetalis in neonates.

Study Location
The University of Uyo Teaching Hospital is located in Uyo the capital of Akwa Ibom State, Nigeria. It was established in 1996 as a state specialist hospital, but was later transformed to a Federal Medical Centre (FMC), in 1997. The hospital was later upgraded to a Teaching Hospital in February, 2007 by the Federal Government of Nigeria. The Hospital is a 500bed tertiary healthcare facility occupying about 43 hectares of land. The institution receives referrals from peripheral healthcare facilities in the state with a population of over 5.7million people in the South-South geopolitical zone of Nigeria.
The Hospital runs a busy Antenatal clinic all through the week and the results used for this study were those of pregnant women booked and attending the antenatal clinic of the University of Uyo Teaching Hospital (UUTH).
In the Obstetrics unit of the hospital, routine screening for the ABO blood group and Rhesus factor is carried out at the booking visit for all pregnant women attending antenatal clinics. Rhesus Negative pregnant women were requested to do the indirect Coomb's test to determine those that are sensitized to the Rhesus D antigen while sensitized pregnant women are managed according to established departmental protocols.

Data Collection
This was a retrospective cross-sectional study conducted at the University of Uyo Teaching Hospital, Uyo, Akwa-Ibom state in South-South Nigeria. The Laboratory registers containing the blood groups of women who attended antenatal clinic from March 2012 to February 2017 were retrieved for this study. Information on the total number of women who attended antenatal clinic, the total number of those who were Rh-Negative mothers, the numbers of those with blood groups A, AB, B and O and the distribution of the Rh status among them were extracted. The folders of the Rh-Negative women were retrieved and further analyzed for isoimmunisation (positive Indirect Coomb`s test). The collated data were analysed using Microsoft excel for windows. Statistical comparison was done using percentages. The results were presented in tables and form the basis for the discussion.

Results
During the study duration, fourteen thousand, three hundred and seven (14,307) women registered for antenatal care at the University of Uyo Teaching Hospital, 629 of them had Rhesus D Negative blood group. This gave a prevalence of 4.40%. (Table 1) The yearly blood group type distribution of all antenatal attendees at the booking clinic from from March 2012 through February 2017 showed an average of 2861 woman booked for antenatal care per year during the 5 year study period and had their blood group checked. (Table 2) The commonest blood group was O with 8232 (57.54%), followed by blood group A, 3416 (23.88%) and blood group B, 2388 (16.69%) while the least common blood group was AB, 271 (1.89%) ( Table 3).
Majority of pregnant women with Rhesus D-negative were of blood group O; 382 (60.78%), followed by group A; 142 (22.58%), and blood group B was 94 (14.49%). AB blood group was the least; 11 (1.70%). (Table 4) Out of the 629 Rhesus negative women, 7 (1.11%) had positive Rh D antibodies at the time of the booking visit while 622 (98.9%) were negative for antibodies. Of these 7 sensitised women, 5 were multigravida, with 2 having had intrauterine foetal deaths (IUFD) late in pregnancy.
Unfortunately, these IUFDs were not investigated because pregnancies and deliveries were managed by the traditional birth attendants (TBA). The other 2, while carrying their first pregnancies past viability, admitted to having terminated 'several' pregnancies without receiving Rh-anti D prophylaxis.

Discussion
The knowledge of blood group and Rhesus factor is important not only in blood transfusion, organ transplantation, forensic pathology and prevention of complications related to Rhesus incompatibility, but in the management of disease conditions and has been found to be associated with dental malocclusion in some populations. [6][7][8][9]15] The prevalence of Rh D negativity among the studied obstetric population was 4.4%. This compared to 4.5%, found in Enugu, South-East Nigeria by Okeke et al, [16] 5.4% by Adewale et al in Oyo, South-West Nigeria, [17] 4.3% by Ezhil et al in Telangana [18] and 4.6% by Kasturi et al in Karnataka [19] Indian provinces. The prevalence of Rh D in our study was however less than 7.1% found in a study carried out in Sokoto, North-West Nigeria, [20] 6.7% and 10% amongst males and females in an Indian study, [21] 19.4% found in an Ethiopian study [22] and 17% among white non-Hispanic donors in America. [23] The highest frequency of the Rh D negativity worldwide was found among the Basques, Spain with a reported prevalence of 47.2%. [24] Studies which showed lower prevalence of Rh negativity relative to ours included a study in the mid-80s which showed a prevalence of 3.3% in Ogbomosho, South-west Nigeria. [25] A lower prevalence of 0.7% was found in Kaduna, Northern Nigeria [26] and 0.3% in Thailand, South-East Asia, [27] while Rhesus negativity was almost nonexistent in a study in Papua New Guinea. [28] From the studies above, there seem to be regional/ethnic differences in the prevalence of the Rhesus gene in Nigeria. A study in Sokoto, north west Nigeria with a high Rhesus D prevalence of 7.1% which involved participants from the major different ethnic Nationalities in Nigeria showed that among the Rh D negative proportion, the Hausas contributed the highest (42.5%), while the Yorubas contributed nothing (0). [20] Another study in Kaduna which had a very low prevalence of 0.7% had the Yorubas contributing the highest proportion (44.73%) among ethnic groups to the Rh Negative population while the Southern minority ethnic groups had the least contributors (3.9%). [26] Even though some of the studies on the prevalence of Rhesus negativity in Nigeria made no mention of the different ethnic groups that contributed to the study, [17] it may however be deduced that the Yoruba ethnic group of Nigeria seem to have the lowest prevalence of the Rhesus blood type against the Hausas who seem to have highest prevalence in Nigeria. [17,20,26] The participants of this study were mainly from the ethnic minorities in South south Nigeria. Apart from the spatial and ethnic/racial variations, the ABO-Rh blood group frequencies had been shown to temporally change in a single population and this may further explain variations in the prevalence of Rhesus negativity in studied populations over time. [29] The yearly booking at the antenatal clinic in the University of Uyo teaching hospital, Uyo ranged between 2382 and 2999, with an average attendance of 2861. The commonest ABO blood group of the women was blood group O, followed by A and B in that order with AB being the least frequent. This pattern of distribution was also found in other studies in Nigeria and beyond. [30][31][32] The ABO group distribution of the Rhesus negative population found mirrored that of the entire antenatal attendees over the fiveyear study period. This was also found by other researchers. [16,33] The low percentage (4.40) of Rhesus negatives in the studied population is an advantage in the light of the prohibitive cost implication of immunoprophylaxis vis-à-vis the very high poverty rate in our population. An Indian study showed a rise in prevalence from 1.8 to 4.6% Rh D negative pregnancy when compared with its prevalence 10 years earlier. [34]. The researchers ascribed the change to possible increased antenatal testing Though, it is known that failure of prophylaxis does occur, most of the women that did not receive the immunoprophylaxis following the previous sensitizing event like delivery or the abortion within the study period had detectable Rh D antibody. It is possible therefore that individual genetic predisposition may be responsible for certain women coming down with isoimmunization even with prophylaxis while others do not despite not receiving immuno-prophylaxis in previous pregnancies/sensitising events. The patients with documented history of adverse effects (IUFD) were not managed in our facility in those pregnancies, hence the scanty details were available about them.

Conclusion
The prevalence of Rhesus negativity is low with an ABO distribution that mirrored that of the antenatal population, hence the overall need for immunoprophylaxis. It is however very necessary to educate and counsel all non-sensitized Rhesus negative women on the need for antenatal and postpartum immuno-prophylaxis as well as sensitised women for management of pregnancies in tertiary health facilities. This is more-so, that once Rhesus immunized mothers remain so for life. There is need for proper public education about this preventable possible sequale of being Rhesus negative.

Limitations of Study
Information was manually extracted an could be subject to error as laboratory data in the University of uyo Teaching hospital during the study period was not computerised.

Funding
The research was not funded by any agency.

Conflict of Interests
The authors declare that they have no competing interests.