{"paper_id":"3c019b73-cf80-4d6b-ad96-9099d88ff3f7","body_text":"This is a preprint and has not been peer reviewed. Data may be preliminary.\nLow Rate of RBC Alloimmunization Among Transfused Children with Sickle Cell Anemia in Malawi Without Pre-Transfusion Screening or Antigen Matching\nAbstract\nRed Blood Cell (RBC) transfusions are an essential component of supportive management for children with sickle cell anemia (SCA) in sub-Saharan Africa. In Malawi, transfusions are matched for ABO/RhD antigens, without screening for alloantibodies or extended antigen matching. We evaluated the prevalence and specificity of RBC alloantibodies in 111 children with SCA and previous transfusions. The alloimmunization rate was 2.7% with antibodies targeting Rh and Kell antigens. This low frequency may reflect similar RBC antigens between donors and recipients in this homogenous population, highlighting the importance of region-specific data and expanded immunohematological profiling to guide transfusion policies in African populations.\nLow Rate of RBC Alloimmunization Among Transfused Children with Sickle Cell Anemia in Malawi Without Pre-Transfusion Screening or Antigen Matching\nApril Evans, MD* (1,2); Kristina Prus, MD* (3,4); Apatsa Matatiyo, MBBS (5); Gugulethu Mapurisa, MBBS, MMED (5); Memory Nonganonga (5); Manley Kamija (5); Kambe Banda-Zgambo, MBBS (5, 6); Timothy Mutafya, MBBS (7); Meffa Misiri (7); Rizine R. Mzikamanda II, MBBS, MMED (8); Katherine D. Westmoreland, MD (5, 9,10); Russell E. Ware, MD, PhD (11,12,13)\n(1) Department of Pediatric Hematology/Oncology, Roger Maris Cancer Center, Fargo, ND (2) Cancer Biology and Immune Therapeutics Group, Sanford Research, Fargo, ND (3) Division of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH (4) Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, OH (5) UNC Project–Malawi, Lilongwe, Malawi (6) Sickle Africa Data Coordinating Center (SADaCC), Division of Human Genetics, University of Cape Town, Cape Town, South Africa (7) Kamuzu Central Hospital, Malawi Ministry of Health, Pediatrics, Lilongwe, Malawi (8) Baylor College of Medicine Children’s Foundation–Malawi, Lilongwe, Malawi (9) Department of Pediatric Hematology Oncology, University of North Carolina, Chapel Hill, NC (10) Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC (11) Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH (12) Division of Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH\n(13) Global Health Center, Cincinnati Children’s Hospital Medical Center, Cincinnati OH\n*Co-First Author\nRed Blood Cell (RBC) transfusions are an essential component of supportive management for children with sickle cell anemia (SCA) in sub-Saharan Africa. In Malawi, transfusions are matched for ABO/RhD antigens, without screening for alloantibodies or extended antigen matching. We evaluated the prevalence and specificity of RBC alloantibodies in 111 children with SCA and previous transfusions. The alloimmunization rate was 2.7% with antibodies targeting Rh and Kell antigens. This low frequency may reflect similar RBC antigens between donors and recipients in this homogenous population, highlighting the importance of region-specific data and expanded immunohematological profiling to guide transfusion policies in African populations.\nINTRODUCTION\nSickle cell anemia (SCA) is the most common inherited hemoglobinopathy in sub-Saharan Africa, affecting millions of children. In Malawi, where medical resources are limited, complications of SCA including severe anemia, infections, and stroke are largely managed by supportive interventions including red blood cell (RBC) transfusions. While transfusions can be lifesaving, they also carry associated risks, notably the development of RBC alloantibodies, which can increase the chance of delayed hemolytic transfusion reactions (DHTR) and complicate future transfusions. 1\nAlloimmunization occurs when a patient’s immune system forms antibodies against foreign (non-self) antigens expressed on donor erythrocytes. Due to the high incidence of alloantibody formation to C, D, E, and K antigens, 2,3 standard transfusion practice for SCA in high income countries includes matching donor blood for ABO group and RhD type; serum screening for IgG alloantibodies; extended matching for Cc, Ee, and K antigens; and occasionally additional minor antigen matching to mitigate DHTR risk. 4,5 Unfortunately, screening and phenotype matching of RBC units are often unavailable in low resource settings, due to cost and limited infrastructure. In practice, transfusion compatibility testing in Malawi usually covers only ABO and RhD matching, without routine antibody screening or donor unit extended phenotype matching.\nAlthough SCA is extremely common in Africa and many children require RBC transfusions, there are relatively few published data on the rates of RBC alloimmunization among pediatric SCA populations in sub-Saharan Africa. Reported rates of alloimmunization vary by design and setting, ranging from 4-9% in cross-sectional studies from Ghana, Nigeria, Uganda and Tanzania. 6–9 However, a recent retrospective longitudinal analysis in Kenya revealed 14% alloimmunization 10 and a prospective study in Côte d’Ivoire documented over 28%. 11 In a recent systematic review of 15 studies conducted in 9 African countries, the average alloimmunization rate was 7.4% (95% confidence interval 5.1-10.0%) but the authors noted the lack of prospective longitudinal data. 12 Across all these studies, the majority of RBC alloantibodies were directed against the Rhesus system, typically D, C, and E along with Kell and other minor antigens. 6–12\nThis study aimed to determine the prevalence of RBC alloantibodies among transfused children with SCA in Malawi, using common serological techniques to characterize the antibodies and inform the development of safer and more contextually appropriate transfusion strategies in the region.\nMETHODS\nThis cross-sectional study was conducted at the Kamuzu Central Hospital Sickle Cell Clinic in Lilongwe, Malawi between January-June 2022. Children <18 years old with confirmed SCA and at least one previous RBC transfusion were eligible. Participants were recruited during routine outpatient visits with written informed consent, plus assent for children >7 years old. Demographic and clinical data included age, sex, lifetime transfusion history (categorized as 1-2, 3-5, 6-10, or >10); and caregiver-reported history of transfusion-related symptoms including fever, pain, chills, rash, trouble breathing, medications or worsening anemia were collected.\nVenous blood samples were collected, with plasma separated and stored at –80C. Alloantibody screening was performed using a commercial 3-cell reagent panel of RBC suspensions. If the screen was positive, an extended 20-cell panel determined antibody specificities. A modified plasma-to-RBC ratio of 1:1 was used to accommodate specimen volume constraints. 13 Antibody identification was performed using standard antibody rule-in and rule-out guidelines.\nRESULTS\nAmong 133 enrolled children with SCA and previous transfusions, 22 were excluded due to insufficient plasma, thus 111 (65 male) samples were analyzed. The average age was 9.3±4.5 years (mean±SD), range 1–18 years. Previous transfusion history included 41% with 1-2 transfusions, 30% with 3-5, 10% with 6-10, and 20% who had received more than 10 transfusions (Table 1). Alloantibody testing was completed for all 111 children.\nOnly three children (2.7%) had detectable RBC alloantibodies. The antibody specificity identified as one with anti-D, one with anti-K, and one with three alloantibodies: anti-D, anti-C, and anti-K.\nThe majority of caregivers (65%) reported post-transfusion symptoms in their children, most often fever, but no child who developed alloantibodies had a recorded or recalled history of a transfusion reaction. No severe or delayed hemolytic reactions consistent with DHTR were recorded.\nDISCUSSION\nIn Malawi, the risks of RBC alloimmunization in children with SCA have not been previously investigated. A low overall alloimmunization rate of 1.1% was reported in the general population and attributed to low RBC antigen diversity including only 3% RhD-negative individuals. 14\nWe found a low rate of RBC alloimmunization (2.7%) among transfused children with SCA. This rate is marginally less than the 5-10% rate in other reported African countries, 6–12 and substantially lower than ~30% rates reported in the United States. 2,3 Several factors may contribute to this discrepancy, most notably age, transfusion frequency, and antigenic compatibility. 2–5,13,15\nAge is relevant as most of our study population was <10 years of age, and longitudinal follow-up might identify a higher cumulative rate. The average transfusion exposure was also modest, with only 30% of the children having >5 lifetime transfusions (Table 1). The most important determinant of alloimmunization may be the diversity within the genes that encode the RBC antigen systems. 1,16,17 The Malawi population is genetically distinct from other African ancestry populations 18, and so genetic similarity between blood donors and recipients in Malawi may reduce the likelihood of mismatched antigens, particularly in the immunogenic Rh and Kell antigen systems. 15,16 In high-income countries, this issue is accentuated by ethnic differences in RBC antigens between predominantly Caucasian blood donors and SCA populations primarily of African descent. 2,3,13 However, even the use of blood from African American donors does not eliminate the alloimmunization rates in US SCA populations, 17 presumably due to the high degree of variant antigens across the African continent.\nDespite RhD-matched transfusions, the presence of anti-D antibodies in two children suggests partial or weak D antigen variants among recipients. These variants are known to occur more frequently in individuals of African ancestry and are difficult or impossible to detect by standard serologic testing, which may lead to alloimmunization even when RhD status appears compatible. 13,14,17,19–23 Similarly, detection of anti-K antibodies, uncommon in African populations due to the low frequency of the K antigen, may point to unrecognized antigenic diversity in the African donor population, potentially influenced by genetic admixture, limited donor typing, and medical tourism. 12 RBC antigen diversity in Malawi remains largely unstudied, although new genotyping array methodology should allow a better understanding of the genetic diversity within populations. 24\nLimitations of this study include a modest sample size, low sample volumes, recall bias of number of transfusions, and a single time point analysis. Bood volumes were limited to avoid further anemia, leading to occasional issues with insufficient samples for alloantibody analysis. The small sample size and single time point analysis could be addressed by future prospective analysis of the entire sickle cell population, which might change the alloimmunization rate.\nIn settings like Malawi, routine serum screening and extended antigen matching are unlikely to be feasible due to high costs, reagent stability, and lack of training, and may not be warranted. Ongoing surveillance is needed, however, since country-specific data should guide transfusion policy; findings from one region may not be generalizable across the continent due to the differences in donor pool, genetic background, transfusion frequency, and healthcare system infrastructure. When resources are limited, targeted strategies focusing on at-risk high transfusion populations may offer a cost-effective means of providing safer RBC transfusions by reducing alloimmunization.\nREFERENCES\n1. Yazdanbakhsh K, Ware RE, Noizat-Pirenne F. Red blood cell alloimmunization in sickle cell disease: pathophysiology, risk factors, and transfusion management. Blood . 2012;120(3):528-537. doi:10.1182/blood-2011-11-327361\n2. Vichinsky EP, Earles A, Johnson RA, Hoag MS, Williams A, Lubin B. Alloimmunization in sickle cell anemia and transfusion of racially unmatched blood. N Engl J Med . 1990;322(23):1617-1621. doi:10.1056/NEJM199006073222301\n3. Rosse WF, Gallagher D, Kinney TR, et al. Transfusion and alloimmunization in sickle cell disease. The Cooperative Study of Sickle Cell Disease. Blood . 1990;76(7):1431-1437.\n4. Lasalle-Williams M, Nuss R, Le T, et al. 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Blood . 2025;146(12):1511-1524. doi:10.1182/blood.2025028902\n| Number of Previous RBC Transfusions | Number of Children | Age (years) ±SD | Alloantibodies N (%) | Antigen Specificity |\n| 1-2 | 45 | 7.6 ± 4.8 | 1 (2) | C,E,K |\n| 3-5 | 33 | 9.6 ± 4.1 | 1 (3) | K |\n| 6-10 | 11 | 9.9 ± 4.5 | 1 (9) | D |\n| >10 | 22 | 10.8 ± 4.1 | 0 (0) | - |\nInformation & Authors\nInformation\nVersion history\nCopyright\nThis work is licensed under a Non Exclusive No Reuse License.\nKeywords\nAuthors\nMetrics & Citations\nMetrics\nArticle Usage\n209views\n143downloads\nCitations\nDownload citation\nApril Evans, Kristina Prus, Apatsa Matatiyo, et al.\nLow Rate of RBC Alloimmunization Among Transfused Children with Sickle Cell Anemia in Malawi Without Pre-Transfusion Screening or Antigen Matching. Authorea. 07 October 2025.\nDOI: https://doi.org/10.22541/au.175984209.95367792/v1\nDOI: https://doi.org/10.22541/au.175984209.95367792/v1\nIf you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download.\nFor more information or tips please see 'Downloading to a citation manager' in the Help menu.","source_license":"CC-BY-4.0","license_restricted":false}