Abstract
Hereditary spherocytosis (HS) is the most common form of hereditary chronic hemolytic anemia. It is caused by mutations in red blood cell (RBC) membrane and cytoskeletal proteins, which compromise membrane integrity, leading to vesiculation. Eventually, this leads to entrapment of poorly deformable spherocytes in the spleen. Splenectomy is a procedure often performed in HS. The clinical benefit results from removing the primary site of destruction, thereby improving RBC survival. But whether changes in RBC properties contribute to the clinical benefit of splenectomy is unknown. In this study we used ektacytometry to investigate the longitudinal effects of splenectomy on RBC properties in five well-characterized HS patients at four different time points and in a case-control cohort of 26 HS patients. Osmotic gradient ektacytometry showed that splenectomy resulted in improved intracellular viscosity (hydration state) whereas total surface area and surface-to-volume ratio remained essentially unchanged. The cell membrane stability test (CMST), which assesses the in vitro response to shear stress, showed that after splenectomy, HS RBCs had partly regained the ability to shed membrane, a property of healthy RBCs, which was confirmed in the case-control cohort. In particular the CMST holds promise as a novel biomarker in HS that reflects RBC membrane health and may be used to asses treatment response in HS.
Highlights
Hereditary spherocytosis (HS) is a heterogeneous group of inherited anemias that originates from defective anchoring of transmembrane proteins to the cytoskeletal network of the red blood cell (RBC)
When RBCs of one HS patient were separated according to density (Supplementary Figure 1A), we noted that the mean corpuscular hemoglobin concentration (MCHC) before splenectomy seemed determined mainly by density fraction 4, containing the most dense RBCs, whereas after splenectomy density fractions 3 and 4 seem to contribute to the MCHC (Supplementary Figure 1B), This implies a more homogeneous RBC population after splenectomy, which was reflected by a more equal distribution of the different fractions after splenectomy (Supplementary Figure 1A)
We focused on cellular properties related to membrane health with the use of two different forms of ektacytometry: osmotic gradient ektacytometry and the cell membrane stability test (CMST)
Summary
Hereditary spherocytosis (HS) is a heterogeneous group of inherited anemias that originates from defective anchoring of transmembrane proteins to the cytoskeletal network of the red blood cell (RBC). The defective anchoring is predominantly caused by a mutation in the genes coding for ankyrin (ANK1), α-spectrin (SPTA1), β-spectrin (SPTB), band-3 (SLC4A1), or protein 4.2 (EPB42) (Perrotta et al, 2008). These mutations compromise the vertical linkages between the lipid bilayer and the cytoskeletal network, leading to destabilization of the membrane, increased. The compromised vertical linkages in HS accelerate the loss of membrane and deformability, leading to premature destruction of RBCs in the spleen. The risks and benefits should be carefully assessed as splenectomy results in a permanently increased risk of infections caused by encapsulated bacteria and long term risk for cardiovascular events (Perrotta et al, 2008; Schilling et al, 2008)
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