Dose-dependent effects of diamide and glutaraldehyde on red blood cell deformability, viscosity, and morphology

Red blood cells (RBC) deformability enables passage through capillaries, ensuring efficient microcirculatory flow and oxygen transport. Impaired deformability contributes to vascular complications and is associated with conditions such as sickle cell anemia, hereditary spherocytosis, and diabetes. Diamide and glutaraldehyde are commonly used in vitro to simulate pathological rigidity, yet their dose-dependent effects on RBC mechanics remain incompletely characterized. This study investigates the effects of diamide (10-200 μM) and glutaraldehyde (8-159,800 μM) on RBC morphology, deformability and viscosity at 20% hematocrit (HCT). Deformability was assessed by ektacytometry, viscosity by microfluidic viscometry, and morphology by defocusing microscopy with sphericity analysis. Osmolality and HCT were also measured to link the mechanical changes observed. Diamide produced a biphasic response: the maximum elongation index (EIₘₐₓ) decreased up to 140 μM, then partially recovered at higher concentrations. This coincided with increased sphericity, reduced cell volume and surface area, decreased HCT from hemolysis, and a non-monotonous viscosity profile. These effects reflect the combined influence of oxidative stress, vesiculation, and altered cell geometry. In contrast, glutaraldehyde induced an abrupt and irreversible loss of deformability at ≥7990 μM, while morphology and osmolality remained stable. HCT values were consistently lower across concentrations, which we attribute to reduced microtube filling efficiency by rigidified cells. Despite near-zero EIₘₐₓ at high concentrations, viscosity changes were modest due to extreme rigidification. These findings show that viscosity is governed not only by deformability but also by morphology, hemolysis and suspension dynamics.