Increased Complement Deposition on Red Blood Cells in Children with Sickle Cell Trait.

Aims: Immune-mediated mechanism, such as deposition of complement (C3b) on erythrocytes leading to enhanced receptor-mediated uptake by macrophages has been proposed to contribute partly to the destruction of non-infected cells leading to anaemia. The extent of complement deposition on RBC (red blood cells) may therefore influence an individual's resistance or susceptibility to severe malarial anaemia. Our objective was to see if RBC of sickle cell trait individuals have increased susceptibility to deposition of complement in vivo. Under oxygenated and deoxygenated conditions, cytofluorometry was used to determine susceptibility of RBC from individuals with normal haemoglobin and those with heterozygous sickle cell trait to complement deposition. Methods: Children aged 0-192 months (n=116) were enrolled in the nested case controlled study and were stratified into HbAS (n=47) and HbAA (n=69). The 47 HbAS individuals were matched to the 69 HbAA individuals of similar age (± 2 months or ± 24 months for those below or more than 192 months, respectively) at a ratio of 1:1 or 1:2. We measured the red cell C3b by flow cytometry under normal and reduced oxygen saturation. Individuals who were positive for malaria were treated and blood was collected when they were free of parasitemia. Analysis of variance was used to identify independent variables associated with the complement C3b positive red cells and Hb level. Results: The mean complement C3b-positive cells for the HbAS was significantly higher than HbAA (P=0.0191). This was also true when this was repeated under deoxygenated conditions (P=0.00065). When the study volunteers were grouped by age cohorts into 0-12, 13-48 and 49-192 months, it was noted that generally; the mean complement C3b positive red cells was higher in the HbAS compared to HbAA but was not statistically significant. Under deoxygenated conditions, the trend was the same. However, between the ages of 49-192 months, the difference was statistically significant. Conclusion: Increased complement C3b deposition on red cells of HbAS cells may predispose the HbAS individuals to increased RBC destruction and therefore protection from severe manifestations of malaria.

Sickle Cell Trait (HbAS) is Associated with Increased Expression of Erythrocyte Complement Regulatory Proteins CR1 and CD55 Levels in Children.

Aims: Erythrocyte complement regulatory proteins, complement receptor 1 (CR1) and decay accelerating factor (CD55) protect red blood cells (RBCs) from complement mediated damage by controlling complement activation cascade and potentially protect RBCs from complement mediated damage that may occur when immune complexes are formed following malaria infection. Given the important role of RBCs in regulation of complement activation, we considered the competence of sickle cell trait RBCs in these functions. Methods: Children (age 0-192 months; n=116) were enrolled in a nested case controlled study conducted in Kombewa Division, Kisumu west District between October and December 2004. Based on hemoglobin (Hb) type, children were stratified into those with HbAS (n=47) and HbAA (n=69). The 47 HbAS individuals were matched to the 69 HbAA individuals of similar age (± 2 months or ± 24 months for those below or more than 192 months, respectively) at a ratio of 1:1 or 1:2. Circulating CR1 levels and CD levels were quantified using a FACScan cytometer under normal and reduced oxygen saturation. Results: The mean CR1 copy numbers per RBC was comparable in the two groups. However, between the ages of 49-192 months, the mean CR1 copy numbers per erythrocyte was significantly higher in children who had HbAS compared to those with HbAA (P=0.0332). The mean CD55 levels were comparable between the two groups but after deoxygenation, the mean CD levels in RBCs of individuals with HbAS was significantly higher than in the HbAA (P=0.011). Conclusion: The mean CR1 and CD55 copy numbers per RBC were comparable between the two groups under normal and reduced oxygen saturation. Beyond the age of 49 months, the CR1 copy numbers was higher in the HbAS compared to HbAA and this was also true for CD55 levels under deoxygenated conditions. Taken together, these results demonstrate that in the younger age groups, the protection afforded by HbAS against severe manifestations of malaria may be due to other factors other than complement regulatory proteins but beyond the age of 49 months, this protection may be partly due to the high CR1 copy numbers in the HbAS individuals.

Red Blood Cell Immune Complex Binding Capacity in Children with Sickle Cell Trait (HbAS) Living in P. falciparum Malaria Holoendemic Region of Western Kenya.

Aims: Malaria infection leads to the formation of circulating immune complexes (CICs) which have been implicated in the pathogenesis of complicated malaria which includes severe malarial anemia. Children with sickle cell trait (HbAS) are less predisposed to getting severe manifestations of malaria. We carried out a study to determine the competence of the red blood cells (RBCs) of children with HbAS to bind immune complexes (ICs) and compared this with normal hemoglobin (HbAA). Methods: Children (aged 0-192 months) were enrolled in a nested case controlled study conducted in Kombewa Division, Kisumu West District, Kenya. Based on hemoglobin (Hb) type, children were stratified into those with HbAS (n=47) and HbAA (n=69). The 47 HbAS individuals were matched to 69 HbAA of similar age. The children were further categorized into three cohorts (0-12, 13-48 and 49-192 months). Immune complex binding capacity (ICBC) was quantified using a FACScan flow cytometer under normal and reduced oxygen saturation. Results: The mean immune complex binding capacity for the HbAS cells was significantly higher than that of HbAA cells (P=0.0191) under normal oxygen saturation or under reduced oxygen saturation (P=0.0050). When a matching variable (UNIANOVA) was done to control for age, gender, the presence or absence of malaria parasitaemia, the binding capacity was again significantly higher for the HbAS than for HbAA under normal oxygen saturation (P=0.025) and under reduced oxygen saturation (P=0.003). The binding capacity was lowest in the 7-12 months age group for both HbAS and HbAA; however, the overall picture showed that HbAS individuals had higher immune complex binding capacity than HbAA in all the age cohorts. Conclusion: These results demonstrate that the protection afforded by HbAS against severe manifestations of malaria may be partly due to higher immune complex binding capacity of the HbAS compared to the HbAA cells. This high binding capacity may lead to the mopping up of ICs formed during malaria attacks and therefore protect these cells from deposition and subsequent destruction.

Association between Sickle Cell Trait and Low Density Parasitaemia in a P. falciparum Malaria Holoendemic Region of Western Kenya.

Aims: The frequency of the mutant gene for sickle cell is widely distributed in the sub-Saharan Africa, the Middle East, and the Indian subcontinent. There is epidemiologic evidence that sickle cell trait confers a survival advantage against malaria and that the selection pressure due to malaria has resulted in high frequencies of the mutant gene in areas of high malaria transmission. We carried out a study to look at the relationship between sickle cell trait, age, haemoglobin level, and malaria parasite density. Methods: We carried out a cross-sectional study between the months of October and December, 2004 in Kombewa Division of Kisumu West District, a P. falciparum malaria holoendemic area with entomological inoculation rates estimated at 31.1 infective bites per person per year. We screened and quantified malaria parasitaemia in participants (age 0 to 45 years n = 342). Haemoglobin electrophoresis was performed on blood from all the participants. Results: In total, 402 participants were screened of which 342 were enrolled. Of these, 280(81.4%) had haemoglobin AA, 60(17.4%) had haemoglobin AS and 2(0.6%) had haemoglobin SS. Those with HbAA and HbAS were included for the analysis bringing the total number to 340 participants. For asymptomatic individuals in the community who displayed no signs of an acute or chronic illness and who were P. falciparum malaria parasite positive; the mean parasite density/µL for HbAS of 4064.0 (95% CI 1858.0 – 6270.0) was significantly lower than that of HbAA 11,067.9 (95% CI 7616.0 – 14520.0) (P = 0.001). Conclusion: The sickle cell carrier status is high (17.4%) in this population and is protective against high density parasitaemia. It is suggested that any malaria intervention strategies should factor in the possibility of sickle cell trait as a confounder to the protective effect of the intervention.