Key contributors to cell-free layer formation: An experimental investigation of hematocrit and shear rate gradient.

The formation of the cell-free layer (CFL) near vessel walls plays a critical role in microcirculatory function, influencing blood rheology, oxygen delivery, and endothelial interactions. While hematocrit (Ht) is a well-established determinant of CFL thickness, the influence of shear-related parameters remains debated due to conflicting findings in the literature. In this study, we systematically quantified the optical CFL thickness (δ) in circular glass microchannels (25-50 μm diameter) under varying hematocrit levels (5-20 %), flow rates, and suspension media (phosphate-buffered saline and plasma). High-resolution microfluidic imaging and micro-particle image velocimetry (μPIV) were used to extract local velocity fields and calculate shear rate gradients (∇γ̇). Rather than treating ∇γ̇ as an imposed variable, we characterize it as a flow-derived descriptor of the local hydrodynamic environment. Across conditions, ∇γ̇ showed stronger correlations with CFL thickness than bulk shear rate. In PBS, increasing ∇γ̇ was associated with reduced CFL thickness, likely due to enhanced shear-induced dispersion. In contrast, in plasma, higher ∇γ̇ values promoted disaggregation of red blood cell (RBC) aggregates and restored hydrodynamic lift, resulting in thicker CFLs. These trends underscore the importance of considering both the suspension medium and spatial shear variations when interpreting RBC behavior. Comparison with prior in vitro, in vivo, and computational studies suggests that discrepancies in reported CFL trends can often be reconciled by accounting for differences in aggregation potential and local shear rate gradients. This work provides a unified experimental framework for interpreting CFL dynamics and highlights ∇γ̇ as a valuable parameter for describing flow-mediated RBC redistribution in the microcirculation.