Sickle cell disease (SCD) is a genetic disorder characterized by haemoglobin S (Hb S) resulting from the inheritance of an abnormal beta-globin chain gene from one or both parents. Globally, 50 million people are affected, with Africa experiencing from 50–90 % of childhood mortality.1 In Ghana, screening of newborns between 1995 and 2004 recruited 177,283 newborns with 3346 having SCD. Annually, SCD makes up about 2 % of 5 million total births per year.2 Screening is to ensure that SCD in children is captured early enough for medical treatment. Meanwhile, in Nigeria, it has been estimated that 24 % of the population has the sickle cell trait (SCT), with an estimated 150,000 babies born with SCD annually. SCD makes up 3 % of the total births while the annual infant mortality rate is approximately 100,000.3

In Africa, blood transfusion remains the most common form of therapy for SCD effectively reducing complications including vaso-occlusive crises, acute pain, and chest pain syndrome by increasing the oxygen-carrying capacity of blood.4 However, there are risks associated with blood transfusions such as blood-borne diseases, and allergic and haemolytic reactions. Chronically transfused patients suffer from iron overload and alloimmunization which is particularly prominent in those suffering from SCD. The production of alloantibodies can affect up to one-third of the SCD population potentially resulting in delayed haemolytic transfusion reactions (DHTR).5 Identifying suitable and compatible blood for a patient with multiple alloantibodies has therefore become a challenge.6

The presence of delayed reactions and the development of antibodies in multi-transfused patients has been implicated in various pathological conditions. It has been proposed that one of the causes of the increased alloimmunization rate in individuals with SCD is chronic inflammation.7

Inflammation arises from an abnormal activation of innate immune responses which can be initiated by the process of haemolysis. This commences chronically in SCD when red blood cells are damaged, releasing various molecules into the peripheral blood, including Hb S and heme (iron compound). It was recently discovered that free heme increases in both SCD and beta thalassaemia, however, the inflammation in SCD is triggered by circulating abnormal haemoglobin.8 The abnormal Hb S binds to Toll-like receptor 4 (TLR4: also known as CD284) expressed on monocytes, a key activator of the innate immune response.9,10 The resulting inflammation has been correlated with mortality and therefore it has been hypothesized that elevated levels of pro-inflammatory markers could predict the buildup of allogeneic antibodies.

It has been observed in a murine model that inflammation plays an important role in RBC alloimmunization.11 This work supported the theory that SCD is characterized by chronic inflammation12 and has led to the hypothesis that inflammation plays a role in the increased rate of alloimmunisation observed in these patients despite little published data. This hypothesis appears reasonable because inflammatory signals activate the immune response and advance the recognition of foreign antigens. The release of cytokines, such as interleukin-6 (IL-6), interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-α) as well as the activation of antigen-presenting cells and tissue damage may lead to initiation of alloimmunization when foreign antigens are introduced during transplantation and transfusion.

Alloimmunization poses a complex challenge with the risk increasing after every additional blood transfusion.13 A study carried out in the United States reported that 50 % of all immunized subjects had multiple antibodies.14–16 Over time many of these became undetectable, potentially challenging future transfusion and putting the patient at risk of a DHTR.17 The most common red cell antigens involved are the Rh, Kell, Kid, Duffy, Lewis, and MNS blood group systems.18,19 Other factors include the recipient's age and sex, number and frequency of transfusions, history of pregnancy, recipient clinical diagnosis and treatment, ethnic differences between recipient and donors and genetic factors related to antigenic responses.16

The overall incidence of post-transfusion alloimmunization in Nigeria varies from 18.7 %20 to lower rates of 8.8 %21 depending on the region where the study took place.

With this background, it has been hypothesized that those with elevated levels of inflammatory markers have a higher risk of alloimmunization and delayed transfusion reactions. Therefore, this study aimed to investigate the association between the increased incidence of alloimmunization in multi-transfused individuals with SCD and chronic inflammatory markers. This study focused on a cohort of individuals with SCD diagnosed at the haematology department of an academic hospital in Nigeria.

This was a cross-sectional study conducted at the Department of Haematology and Immunology, Obafemi Awolowo University Teaching Hospital, Nigeria. The complete blood count (CBC), differential count, inflammatory markers, and blood group antigen profiles of patients with SCD (SCD group) who received two or more doses of blood were compared to a group of individuals without the disease (Control group) but who also received blood transfusions.

Correlation analysis showed weak positive correlations between alloimmunization and two of the pro-inflammatory markers, CRP, and TNF-α. In contrast, very weak negative correlations between alloimmunization and two other inflammatory markers, IL-1β and IL6, were observed. These however were not statistically significant (Table 5).

This cross-sectional study aimed to explain the complex relationship between inflammation and alloimmunization in individuals with SCD. It highlights the increased transfusion frequency and transfusion reactions in these patients. The participants with SCD developed allogeneic antibodies which were mostly anti-Kell, Jka and Fya while the non-SCD group developed anti-Kell and anti-M antibodies. Despite the individuals with SCD having significantly elevated levels of pro-inflammatory markers (TNF-α and CRP), no significant correlation was detected with the rate of alloimmunization. This suggests that while inflammation is prevalent in patients with SCD, it does not directly predict alloimmunization.25

These findings align with previous research by Kangiwa et al.20 which reported similar alloimmunization patterns but at higher rates. Differences in sample size, subject age, regional practices, and transfusion protocols likely contribute to the variance in alloimmunization rates. This study focused on young adults in the Western region of Nigeria, while Kangiwa et al.20 included both adults and children in the Eastern region, indicating possible regional differences in antigen prevalence and healthcare practices.

Other studies26,27–29 corroborate these findings, highlighting the clinically significant increases in Rhesus and Kell antibodies. The alloimmunization rate in Nigeria is higher compared to other African countries like Ghana30 Uganda31 and Tanzania.17

These differences might be explained by variations in the genetic backgrounds between populations which can influence immune responses. For instance, the prevalence of certain antigens like Kell, Jka, and Fya might vary across different ethnic groups, affecting the likelihood of developing corresponding antibodies. Likewise, variations in blood transfusion protocols and donor screening processes between different regions and hospitals can impact alloimmunization rates and the types of antibodies formed.

At our institution, blood units are prophylactically phenotype-matched between the recipient and the donor using ABO and Rhesus grouping and when compatible given to the recipient without necessarily carrying out antigen testing. However, in this current study, an extended antigen testing panel was performed.

The immunogenicity of antigens may also significantly influence the likelihood of antibody development. Antigens like Kell, Jka, and Fya, which were elevated in the SCD group, are known to be highly immunogenic, leading to a higher prevalence of corresponding antibodies in transfused patients. The Duffy antigen, specifically Fya, is particularly immunogenic and prevalent in African populations, making it a common target for alloimmunization in individuals with SCD.32,33

In this current study, individuals with SCD had significantly higher levels of the proinflammatory proteins TNF-α and CRP while IL-6 and IL1 levels were similar to the Control group. The increase in inflammatory proteins has been reported by previous studies34 and it has been proposed that these proteins could be used as clinical biomarkers. For example, CRP has been associated with acute chest pain (ACP) and vaso-occlusive crises.35 This has been confirmed by others whose findings were consistent with this study.36,37

TNF-α is a cytokine with several properties including the activation of endothelial cells and leucocytes. The action of macrophages and the chemotaxis of inflammatory cells has been implicated in the pathogenesis of various acute and chronic states such as sepsis, chronic infections, and inflammatory conditions. TNF-α plays an essential role in the synthesis of protein and the expression of adhesion molecules in vascular endothelial cells.38,39 In SCD, this cytokine has been proposed as a risk factor for the occurrence of painful crises, as well as being involved in the occlusion of the microcirculation.40,41

High white blood cell counts and neutrophils are also associated with inflammation and during infections, neutrophils are the first cells to respond. Activated neutrophils release enzymes, such as reactive oxygen species, proteases and myeloperoxidase, which combat foreign organisms at the infection site.42 These enzymes are also involved in several inflammatory processes.42 When adhesion takes place, chemokines and cytokines are produced which go on to stimulate dendritic cells resulting in the presentation of antigens to memory CD4-positive T cells as well as to naïve CD8-positive T cells which consequently leads to the activation of the adaptive immune response.43

Several other studies have reported elevated platelet counts in individuals with SCD.44–46 Increased platelets in this study could contribute to the chronic and acute complications of SCD by promoting molecular and diverse cellular events within the microcirculation that eventually lead to vaso-occlusive and vascular injury.

Despite the increase in inflammatory markers, no significant correlation between the rate of alloimmunization and any of the inflammatory markers could be detected, which was similar to a previous study by Tatari-Calderone et al.25 Their study, conducted in Washington, DC, USA, involved 83 children with SCD who received multiple red blood cell transfusions for both the prevention and treatment of disease-related complications. The levels of cytokines were correlated with the development of anti-RBC antibodies within the seven-year period post-recruitment and demonstrated that twelve subjects had significantly elevated levels of all cytokines, both pro-inflammatory and anti-inflammatory. Interestingly, higher levels of cytokines were also found in the patients without anti-RBC allo- or auto-antibodies. Therefore, it was concluded that high cytokine levels were not indicative of alloantibody development and that the increased concentration of multiple cytokines is not a biomarker of either the presence of or susceptibility to the development of RBC alloimmunization.

Several other studies have reported increased inflammation and innate immune activation in individuals with SCD47–49 however, the pattern of cytokine expression varies.50,51 High plasma levels of TNF-α have been reported while others have suggested that reduced production of IFN-γ was the first evidence of the onset of DHTR in individuals with SCD.52

Researchers have shown that various factors, aside from inflammation can influence the development of alloimmunization in SCD. These factors include iron overload, haemolysis, delayed haemolytic transfusion reactions, pregnancy, haemolytic disease of the newborn, infection, genetic factors, the antigenic immunogenicity of RBCs, recipient exposure to foreign donor antigens, the immunological status of the recipient, age at first transfusion, and the duration of transfusions. A further factor which could play a role is differences in the human leukocyte antigen alleles. These findings all warrant future research.53,54

This study was conducted in only one region of Nigeria and therefore the results cannot be applied to the general Nigerian population or other countries within Africa. Regional genetic variations, environmental factors, and healthcare practices can influence the results, thereby limiting the broader applicability of the conclusions. A further limitation was the relatively low number of participants. A larger sample size would have provided more robust data and enhanced the statistical power of the study, allowing for more definitive conclusions. Moreover, only four inflammatory markers were analysed which may have overlooked other relevant biomarkers which could provide additional insights into the conditions being studied.

Despite these limitations, the results indicate that SCD individuals in this region of Nigeria have high rates of alloimmunization and elevated levels of anti-Kell, Jka, and Fya antibodies, along with inflammatory markers, TNF-α and CRP, compared to those without SCD. However, no significant correlation was found between the inflammatory markers and alloimmunization. This study underscores the multifactorial nature of alloimmunization in SCD and the importance of considering genetic, regional, and procedural factors to optimize transfusion practices. Further research is needed to explore these differences and develop strategies to reduce alloimmunization risks in individuals with SCD.

[24].