The Prevalence of Red Cell Antigens and Antibodies in the Malawi Population

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1 Report of the Research Study on The Prevalence of Red Cell Antigens and Antibodies in the Malawi Population M baya B*, Mfune T*, Mogombo E*, Mphalalo A*,Ndhlovu D*, Knight R C** *Malawi Blood Transfusion Service, PO Box 2681, Blantyre, Malawi **National Blood Service, Colindale Avenue, London, England All communication to Dr B. M baya Malawi Blood Transfusion Service Galaxy House P.O Box 2681 Blantyre Tel: +265 (1 ) Fax: +265 (1) September

2 Summary As there were no reliable data for the prevalence of red cell alloantibodies or antigens in the population of Malawi, a study was conducted to screen patients for the presence of antibodies and to type them for ABO, RhD, C, c, E, e and K antigens. Five hundred donors were also tested for these antigens plus Fy a, Fy b, Jk a, Jk b, S, s. Red cell antibodies were identified in 11 patients [1.1%]; 2 were anti-d, 2 anti-s, 1 anti-le a+b and 6 anti-m, 4 of which were found in non-transfused males suggesting they might be naturally acquired. The antigen frequencies found were similar to those in literature but with 97.2% of donors being Fy(a-b-). All patients tested were K negative and only 3 donors were found to be K positive, one being Caucasian. Approximately 3.5% of Malawians are RhD negative, lower than the usual 8% quoted for Black Africans. The incidence of the R1 gene is also lower than reported figures. The frequencies found show a high level of red cell antigen homogeneity among Malawians except for the S, RhE and RhC antigens. These data confirm the assumption made that pre-transfusion and antenatal antibody screening is not necessary. As haemolytic disease of the newborn (HDN) is rare in Malawi, more work is needed to find the real incidence of this condition. Background The prevalence of red cell alloantibodies varies greatly from population to population and is dependent on many factors such as the blood group heterogeneity of the population, the presence of different ethnic groups with different blood group frequencies and the number of patients needing multiple transfusions. Except for ABO and RhD, there are no recent data on the frequency of other red cell antigens in the Malawi population. Current developments in the health sector in the country mean that the absence of this data can no longer be ignored. Until 2004, when the Malawi Blood Transfusion Service (MBTS) was established, all the blood used for transfusion in the country was from family replacement blood donors with severe blood shortages at most times of the year. Since its establishment, the MBTS has made blood from voluntary non-remunerated blood donors more readily available. The majority of transfusions in Malawi are for children who become anaemic as a result of malaria. As most of them are being saved, some will require further transfusions in their lifetime. Due to the improved availability of blood, patients requiring multiple transfusions are receiving them and living longer. Furthermore, there are advanced plans to establish a cancer centre in the country that will increase the number of patients requiring multiple blood transfusions as part of their care. These trends mean that red cell antibody formation may become a problem depending on the red cell heterogeneity of the Malawi population. Pre-transfusion testing in most hospital blood banks is still limited to a saline major crossmatch and not the recommended indirect antiglobulin testing (IAT). Clinically significant IgG antibodies will not be detected using only the saline crossmatch method. Therefore, it was thought necessary to establish the baseline prevalence of red cell alloantibodies and the frequency of the major blood group antigens in patients in hospitals in Malawi and among blood donors. This would enable the MBTS to plan its strategy for blood collection and testing, as well as for giving expert support to Hospital Blood Banks. This was well demonstrated by Lin-Chu et al in Taiwan 1 where, having done such studies, they decided on a simple, cost effective compatibility testing strategy rather than following established North American or European standards. 2

3 Methods To establish the prevalence of red cell alloantibodies, adult patients, including antenatal women, were enrolled from hospitals across the country. They were tested for the presence of alloantibodies using DiaMed gel technology. Those samples found to be screen positive were further tested to establish the specificity of the antibodies involved. To establish the frequency of the Rh and K antigens in Malawians the same patient samples used to test for antibodies were typed for the Rh C, c, E, e, and K antigens in addition to ABO and RhD. In addition 500 donors attending MBTS sessions from different parts of Malawi were tested to establish the frequency of ABO, RhD, C, c, E, e, K, Fy a, Fy b, S, s, Jk a, and Jk b antigens. The study was approved by the National Health Sciences Research Council of Malawi. Only patients who gave informed consent were enrolled into the study. Each patient sample was identified by, as a minimum, name, age, gender and date of collection. Each sample was then given a laboratory ID number; the data stored electronically was ID number, age and gender and, where applicable, previous transfusion and pregnancy data. All patient and donor data remains confidential. Although any findings in this study would not have any direct health consequences for the patient, it was agreed that patients who wished to know their test results be informed and that those who had alloantibodies be given an antibody card. No additional consent was required from donors as they had already consented to their blood being tested for blood groups. The testing was performed using DiaMed gel-card technology in accordance with the manufacturer s protocols. Reagent red cells for antibody screening [2-cell screen] and a 10-cell antibody identification panel, suitable for use in the DiaMed system were supplied by the National Blood Service, UK. Each batch of tests was validated using standard positive and negative controls. Where possible, samples were tested within 7 days of collection. DiaMed gel-card technology is simple to use and although more expensive than the reagents required for tube techniques, it proved well suited to the type of testing needed for this project. The laboratory work and data handling were performed by two senior technicians from the MBTS Blantyre laboratories having been trained to use the DiaMed technology. Results patients were enrolled in the study and 500 blood donor specimens were analysed. The general characteristics of the patients in the study are given in table 1. Table 1: General Characteristics of Patients in the Study No. of Parity Age Sex Subjects (female patients) Previous Transfusions Range Mean M F M:F ratio Range Mean No. Range Mean no. of transfusions ± : ± ±1.0 All the test data were considered acceptable except for one batch of 12 samples for anti-s, -s and anti-jk a, -Jk b testing where, at the beginning of the project some technical difficulties were encountered. The results obtained from 500 donors tested for ABO, RhD, C, c, E, e; K, Fy a, Fy b, Jk a, Jk b, S and s antigens are given in tables 2 and 3. 3

4 The results obtained from testing patients for ABO, RhD, C, c, E, e and K antigens are given in table 3. The tables show the frequency of the various antigens in the donor and patient populations of Malawi. For easy comparison, the last two columns of tables 2 and 3 show the frequencies of these antigens as reported in literature. Table 2: ABO and RhD antigen frequencies (%) Blood Donors (%) Patients (%) US Blacks (%)* US Caucasian (%)* A AB B O RhD positive *US Black & Caucasian figures from Reid ME & Lomas-Francis C 2 Table 3: Antigen frequencies of donors and patients (%) Antigen Donors (%) Patients (%) US Blacks (%)* US Caucasian (%)* D C c E e K Fy a Fy b Fy(a+b-) Not tested Fy(a+b+) Fy(a-b+) Fy(a-b-) Jk a Not tested Jk b S s 90.0 Not tested S-s * US Black & Caucasian figures from Reid ME & Lomas-Francis C 2 Table 4 gives the probable Rh genotype based on the test results. 4

5 Table 4: Probable Rh genotypes Rh type Donors Patients US Blacks* US Caucasian* Ro R 1 r / R 1 R o R 2 r / R 2 R o R 1 R R 1 R R 2 R rr r l r R ll r * figures from Mourant et al 3. The distribution of the human blood groups. Of the patient samples screened for antibodies, 13 [1.3%] were found to be positive. Antibody specificity was determined in 11 samples. There were two examples of anti-d, one only weakly reactive; six examples of anti-m, two anti-s, and one anti-le a+b. In the other two samples no specificity was determined. Details are given in table 5. Table 5: Patients with alloantibodies Antibody Gender Age Transfusions Parity 1 Anti-D [weak] F Anti-D F Anti-S F Anti-S F Anti-M M 28 0 N/A 6 Anti-M M 22 0 N/A 7 Anti-M M 25 0 N/A 8 Anti-M M 39 0 N/A 9 Anti-M F Anti-M F Anti-Le a+b F DISCUSSION Antibodies The patient population is young (mean age 30) and this reflects the young Malawi population as the life expectancy is only 36 years. The frequency of alloantibodies in this study is low, 1.1%. Considering that 10% of the patients in the study had been previously transfused and many had multiple pregnancies (refer to table 1), the alloimmunisation rate seems low. This is probably due to the red cell homogeneity of the population. In countries with a more diverse ethnically mixed population, alloimmunisation rates are higher. The predominant antibody found was anti-m [six examples]; however the DiaMed technology used does have a propensity for detecting anti-m. Antibodies of this specificity often are a mixture of IgM and IgG antibodies and many do not react in standard IAT tube methods and are therefore considered to be clinically non-significant [Redman & Malde] 4. Four of these antibodies were found in un-transfused males aged 22 to 39 years and therefore might be naturally acquired. Two examples were found in females neither of whom had been transfused but both had been pregnant. As DiaMed gels are not used in hospitals in Malawi for compatibility testing it is unlikely that anti- M will cause any significant problems. 5

6 Because of the benign nature of anti-m and anti-n antibodies, typing for these two antigens was not included in the initial study although it is now planned to test a small cohort of donors for M and N antigens. With the incidence of RhD negative individuals being so low, approximately 3.5%, it is perhaps not too surprising that anti-d was only found in two patients, one example of which was only weakly reactive. Both patients were female, one had had 2 pregnancies the other 4 pregnancies but neither had been transfused. Anecdotal evidence is that HDN is very rare or not recognised as there are many other causes of neonatal anaemia and jaundice. The other clinically significant antibody found was anti-s, two examples. The S antigen is found in 36.6%. As such, the chances that someone who is S negative may be exposed to the S antigen as a result of pregnancy or transfusion are quite high. In Malawi, pre-transfusion antibody screening is not performed, and indeed not all hospital laboratories yet use an IAT in their crossmatch let alone being able to identify antibody specificity. However, if a serological incompatibility was caused by anti-s, finding a suitable compatible unit would not be too difficult by crossmatching more units of blood by an IAT. One example of anti-le a+b was identified in a 21 year old female in a surgical ward; she had not been transfused but had had two pregnancies. The frequency of Lewis antigens was not determined in this study as the antibodies are considered to be mostly not clinically significant but reported frequencies for Le(a-b-) individuals in the US Black population is 23%. Antigens There is some variation between the data for donors and patients but the donors are a self selected group and will include some non-black Africans whereas the patient cohort was all local Malawians. Samples from both cohorts were taken across the country to reduce any regional bias and data analysed from each of the three regions showed little difference. Blood group antigen frequencies quoted for Africans are often based on data from Afro- Caribbean populations in the USA that do not reflect the regional differences found throughout the continent of Africa. Other data are now quite old and often done on small groups or tribes mainly for anthropological studies. The incidence of R o [R o R o ] at approximately 66% is higher than the 42.2% reported for US Blacks and the 41.8% found among the UK Black population. The R 1 gene is less common than generally reported but the R 2 gene appears to be similar to that normally quoted for black populations. All the patients tested were K negative and only three of the donors were positive; one was Caucasian while the other two Black African. The family history of these two individuals is unknown but there could have been some Caucasian blood in their lineage or from one of the Black African groups in which a higher incidence of K+ has been reported; 10.3% in a small sample of 106 Bantus and 5.2% among 201 Hottentots were K+ [quoted by Mourant et al] 3. The 100% K negative figure for patients is in agreement with a report from Ghana [Narter-Olaga] 6 in which donors tested were all K negative. Because the incidence of the K antigen is so very low the likelihood of anti-k being found in the general hospital population in Malawi is very small whereas in the UK, anti-k is the commonest antibody found after anti-e. [Redman et al] 5 The incidence of Fy(a-b-) at 97.6% is higher than the 68% usually quoted for Black Africans and the 66.4% found in the black population in London, but similar to earlier reported frequencies in Tanzania and Zambia quoted by Mourant et al. 3 6

7 The Duffy antigen on red cells acts as the receptor for Plasmodium vivax so the absence of the Duffy carrier protein in Fy(a-b-) make these cells resistant to invasion by this parasite. Data from the local Malaria Research Project shows that P. vivax is very rarely found in Malawi whereas P. falciparium is one of the major causes of fatality in children. In cases of malaria studied during January to July 2008 in Malawi, only one case caused by P. vivax was found [Heyderman personal communication] 7. The incidence of S and s antigens and for the phenotype S-s- [0.8%] is not too dissimilar to other published data for Black Africans. Most individuals who are S-s- are also negative for the U antigen but the frequency of U- is variable within Black African populations varying from 0.17% to 1.4%, averaging at about 1%. The Jk types are consistent with other data on Black African populations. CONCLUSION The frequency of blood group antibodies and antigens found in this study will help with future planning of the blood transfusion services in Malawi. This study confirms the assertion that the prevalence of alloantibodies in the current population of Malawi is low. As such, there should not be many problems in finding compatible blood. Apart from the S, RhE and RhC, there is a high level of homogeneity in the red cell antigen distribution among the rest of the antigens tested for in this study. Considering the low antigenicity of the three red cell antigens where there is a high degree of heterogeneity, it is anticipated that red cell alloimmunisation will not be a major problem for Malawi, in the near future. At the same time, it also means that should a patient develop antibodies against antigens found in the majority of the population [eg anti-u], it will be difficult to find a suitable blood donor for such patients. At present, because of this low incidence of significant antibodies in patients, any further typing of donors for antigens other than ABO and RhD is not necessary. These data also suggest that pre-transfusion antibody screening is not required but red cells for transfusion should be crossmatched by IAT. If an incompatibility is found then the pragmatic approach of crossmatching further units to find sufficient compatible blood is acceptable. Samples can subsequently be referred to the central MBTS laboratory for antibody identification but transfusion should not be withheld awaiting these results. The low RhD negative rate, (about 3.5%) mean that it is difficult to have sufficient amounts of RhD negative blood for all RhD negative patients. It also means that most RhD negative women will have RhD positive partners and HDN may be a big problem for these women. Anti-D immunoglobulins are not available in hospitals in Malawi. There is need for further research on RhD and HDN in the country. To help improve transfusion practice in Malawi there is an active training programme, led by MBTS, to assist hospital blood banks in good transfusion laboratory practice that will underpin this approach to providing safe and compatible blood. This training is supported by a grant from Centres for Disease Control and Prevention (CDC) of the USA through its Malawi Office. Acknowledgements Thanks to Mr Alan Lawson, DiaMed-GB Ltd. for kindly supplying the reagents used for this project. Thanks also to all hospital nurses and laboratory technicians who worked as research assistants in this study. 7

8 References 1. Lin-Chu M, Broadberry RE, Chang FJ. The distribution of blood group antigens and alloantibodies among Chinese in Taiwan. Transfusion 1988; 28: Reid ME, Lomas-Francis C. The blood group antigen Facts Book. 2 nd Edition, 2004; Elsevier Ltd 3. Mourant AE, Kopec AC, Domaniewska-Sobczak K. The distribution of the human blood groups and other polymorphisms. 1976, Oxford University Press 4. Redman M, Malde R. Personal communication 5. Redman R, Regan F, Contreras C. A prospective study of the incidence of red cell alloimmunisation following transfusion. Vox Sang 1996; 71: Narter-Olaga E. Personal communication 7. Heyderman R personal communication 8

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