Severity-Dependent Modulation of Red Blood Cell Aging Patterns in Preeclampsia: Insights from Calorimetry and Atomic Force Microscopy

Preeclampsia (PE) is associated with systemic oxidative stress and vascular dysfunction, yet its effects on red blood cell (RBC) stability and mechanics remain incompletely understood. Here, we investigate the structural and nanomechanical alterations of RBCs in third-trimester pregnancies complicated by non-severe and severe PE, compared with normotensive controls. RBCs are analyzed using differential scanning calorimetry (DSC) to assess protein thermal stability and atomic force microscopy (AFM) to determine membrane elasticity (Young's modulus) during in vitro aging. Linear mixed-effects models are applied to evaluate the effects of disease severity, storage time, and their (group × storage time) interaction. DSC reveals that Band 3 and hemoglobin exhibited pronounced destabilization in PE, with severe cases showing earlier and larger reductions in transition temperatures and heat capacities, indicative of disrupted membrane-cytoskeletal interactions. AFM confirms that these molecular changes translate into functional consequences: control and non-severe PE RBCs show physiological softening over time, whereas severe PE RBCs undergo pathological stiffening. Statistical modeling demonstrates strong time, group, and interaction effects for both thermodynamic and mechanical parameters. Together, these findings identify the Band 3-hemoglobin macrocomplex as a primary target of PE-induced RBC alterations and suggest that combined thermodynamic-nanomechanical profiling can serve as a sensitive approach to detect early subclinical RBC damage not detectable by routine hematological tests.