Insights into determinants of spleen injury in sickle cell anemia

Abstract

Spleen dysfunction is central to morbidity and mortality in children with sickle cell anemia (SCA). The initiation and determinants of spleen injury, including acute splenic sequestration (ASS) have not been established. We investigated splenic function longitudinally in a cohort of 57 infants with SCA enrolled at 3 to 6 months of age and followed up to 24 months of age and explored the respective contribution of decreased red blood cell (RBC) deformability and increased RBC adhesion on splenic injury, including ASS. Spleen function was evaluated by sequential ^99mTc heated RBC spleen scintigraphy and high-throughput quantification of RBCs with Howell-Jolly bodies (HJBs). At 6 and 18 months of age, spleen filtration function was decreased in 32% and 50% of infants, respectively, whereas the median %HJB-RBCs rose significantly (from 0.3% to 0.74%). An excellent correlation was established between %HJB-RBCs and spleen scintigraphy results. RBC adhesion to laminin and endothelial cells increased with time. Adhesion to endothelial cells negatively correlated with splenic function. Irreversibly sickled cells (ISCs), used as a surrogate marker of impaired deformability, were detected at enrollment and increased significantly at 18 months. %ISCs correlated positively with %HJB-RBCs and negatively with splenic uptake, indicating a relationship between their presence in the circulation and spleen dysfunction. In the subgroup of 8 infants who subsequently experienced ASS, %ISCs at enrollment were significantly higher compared with the asymptomatic group, suggesting a major role of impaired deformability in ASS. Higher levels of %HJB-RBCs were observed after the occurrence of ASS, demonstrating its negative impact on splenic function.

Graphical abstract

Figure 1.

HJB-RBCs and ISCs in children with SCA. (A) %HJB-RBCs determined by IFC in 45 children at 3 to 6 and 18 months. ****P < .0001, Wilcoxon paired test. (B) Distribution of %HJB-RBCs with respect to splenic uptake as measured by ^99mTc heated RBCs spleen scintigraphy. Splenic uptake of 100% to 150% (n = 15), 75% to 100% (n = 4), 50% to 75% (n = 1), 25% to 50% (n = 2), and 0% to 25% (n = 5). (C) %ISCs determined by IFC in 45 children at 3 to 6 and 18 months. ****P < .0001, Wilcoxon paired test. (D) Spearman correlation between %ISCs and %HJB-RBCs at 3 to 6 (red dots) and 18 months (blue dots). n = 99; R² = 0.69; ***P < .001.

Figure 2.

Lu/BCAM expression and mediated RBC adhesion. (A) Microscopic images of RBCs adhering to TrHBMEC monolayers and laminin 521-coated microchannels at 3 to 6 and 24 months. Original magnification ×20. (B-C) The amount of adherent RBCs/mm² on TrHBMEC-coated channels (n = 15) (B) and laminin-coated channels (n = 15) (C) at 3 to 6, 12, 18, and 24 months. *P < .05, **P < .005, ***P < .001, Wilcoxon test. (D) Mean fluorescence intensity of Lu/BCAM on mature RBCs. ****P < .0001, Wilcoxon test.

Figure 3.

RBC adhesion and HJB-RBCs in children with SCA. Spearman correlation between the amount of adherent RBCs/mm² on laminin (A) and TrHBMEC and %HJB-RBCs (B). At 3 to 6 (red dots) and 18 months (blue dots). n = 28; R² = 0.43; ***P < .001.

Figure 4.

HJB-RBCs and ISCs in asymptomatic and ASS-affected infants. Comparison of %HJB-RBCs (A) and %ISCs (B) at 3 to 6 and 18 months in asymptomatic (n = 22) and ASS infants (n = 7, for whom samples were available for analysis). **P < .005, Wilcoxon test.