Development of a hemolysis filter system for the selective removal of free hemoglobin, haem and iron from blood envisaged for use in extracorporeal circuits.

Hemolysis, the rupture of red blood cells, releases hemoglobin, haem, and redox-active iron into the bloodstream. When the body's scavenging capacity is overwhelmed, these species can exert deleterious effects, as seen in hemolytic disorders. Hemolysis can also occur in extracorporeal circuits due to mechanical forces acting on blood during circulation. To mitigate these effects, we developed a filter designed for integration into extracorporeal circuits that is designed to capture hemolysis-associated byproducts using immobilised ligands. The filter prototype comprises glyoxal-functionalized agarose beads covalently immobilized with three binding agents: haptoglobin (Hp, binds cell-free hemoglobin [cfHb]), human serum albumin (HSA, binds haem), and desferrioxamine (DFO, binds free iron). Ligand immobilization was optimized to achieve strong covalent attachment to the agarose matrix. Optimized immobilization produced high ligand loading per milliliter of beads (mean ± SD): Hp 74.4 ± 11.7 mg/mL, HSA 84.6 ± 8.2 mg/mL, and DFO 43.3 ± 4.8 mg/mL. In vitro studies showed removal capacities of 12.7 ± 0.2 mg/mL for cfHb, 2708.5 ± 18.5 µg/mL for haem, and 309.7 ± 13.7 µg/mL for iron. The system retained binding activity in plasma and hemolyzed whole blood, and no increase in TAT or D-dimer was detected under the tested ex vivo conditions. These findings demonstrate the potential of this affinity-based filtration system to reduce hemolysis-associated complications in extracorporeal circulation.