(455h) The Influence of Red Blood Cell Deformability and Hematocrit on Vascular-Targeted Carrier and White Blood Cell Adhesion from Blood Flow

The Influence of Red Blood Cell Deformability and Hematocrit on Vascular-Targeted Carrier and White Blood Cell Adhesion from Blood Flow

Margaret Fish¹ and Omolola Eniola-Adefeso¹

(1) Chemical Engineering, University of Michigan, Ann Arbor, MI

Introduction: Blood is a highly concentrated suspension of red blood cells (RBCs), white blood cells (WBCs), platelets, and plasma. The RBC core of blood flow develops due to wall-induced migration and the cellular dynamics of collision, which promotes margination of WBCs and platelets to the vascular wall.^1,2 Margination, by definition, is the localization and adhesion to a vascular wall from flow conditions. In our previous work, we have shown interaction of vascular-targeted carriers (VTCs) with the vascular wall is greatly impacted by RBCs.³However, the physics of RBCs in flow is altered in many diseases, which has the potential to alter the ability of WBCs and VTC to marginate to the vascular wall. VTCs must be able to successfully exit bulk blood flow and come in contact with the vessel wall in order to achieve their apecific vascular adhesion. Some diseases, such as sickle cell anemia and malaria, alter the mechanical properties of RBCs and produce side effects including vessel occlusions, intense pain, and strokes. Based on previous work showing that physical cell properties such as size, shape, and modulus are important in cellular distributions, we experimentally investigate how altering RBC rigidity affects the distribution of cells and VTCs in flow. In a related project, the increased hematocrit (Hct) in diseases such as polycythemia vera greatly alters hemodynamics and has the potential to affect WBC and VTC function at the vascular wall.

The purpose of this project is to elucidate the effect that rigidified RBCs and increased Hct have on both VTC and WBC adhesion to an inflamed endothelium in vitro.

Materials and Methods: Blood was obtained from healthy human donors and the RBCs were isolated by dextran sedimentation. The deformability of RBC membranes was altered by incubating in RBCs varying concentrations of tert-Butyl hydroperoxide (tBHP) to induce lipid peroxidation and subsequent crosslinking with membrane proteins. The elongation index for RBCs treated with a range of tBHP concentrations was measured by ektacytometry.

Selectin-targeted, polystyrene particles and WBCs were tested for their adhesion efficiency in in vitro parallel plate flow chamber (PPFC) assays. Healthy human blood mixed with artificially rigidified RBCs, while controlling for total Hct, was perfused over a monolayer of IL-1β activated human umbilical vein endothelial cells (HUVEC). Adhesion of VTCs and WBCs from laminar blood flows ranging in wall shear rates (100-1000 s^-1) were investigated. The percent of rigid RBCs, out of total RBCs, was varied from 0% to 100% when investigating both VTC and WBC adhesion. Additionally, the effect of the rigidity of the rigid RBCs, controlled by the concentration of tBHP, was investigated on VTC and WBC adhesion. The number of adherent particles or WBCs under various flow conditions was imaged/quantified using optical microscopy.

The flow distribution of RBCs with different mechanical properties was investigated using confocal microscopy. The particle/blood mixture was flowed through a microchannel, which was placed under 20x magnification on an upright confocal microscope.  Focal planes were set in the CFL, starting from the top of the channel, and the number of stained RBCs, fluorescent particles, or stained WBCs were quantified in 10-µm increments to determine where each cell type or VTC flows.

The effect of increased Hct on WBC binding in vitro was investigated by adding isolated RBCs to whole blood. Additional WBCs were added to match WBC concentrations between bloods of differing Hcts. WBC concentration for each donor was counted using a hemacytometer. Further experiments investigate the effect of increasing Hct on VTC binding from whole blood in vitro, with the same aforementioned PPFC assay method.   

Results and Discussion: Preliminary results from in vitro PPFC assays show that rigidified RBCs affect the adhesion of VTCs and WBCs to an inflamed endothelium in vitro. The effect in adhesion is not linear as the percentage of rigidified RBCs increases linearly, which highlights the complexities that exist in blood flow. More subtle changes in adhesion were observed when less rigidified RBCs were used in the PPFC assay. Additionally, the distribution of healthy RBCs is distinct from rigidified RBCs in bulk flow in vitro.

Increasing the Hct of blood significantly decreased adhesion of both WBCs and VTCs to an inflamed endothelium in vitro. In general, the 80% Hct condition produced a larger drop in adhesion than the 60% Hct, as compared to the 40% Hct control. This is likely due to the decrease in size of the RBC-free layer, which leaves less space for margination and increases the number of disruptive collisions between RBCs, WBCs, and VTCs near the vascular wall.   

Conclusions and Impact:

This work investigates the transport step of WBCs from bulk blood flow to the vessel wall in the context of altered Hct levels and rigidified RBCs. This project demonstrates that both RBC rigidity and Hct affect the adhesion efficiency of WBCs and VTCs in vitro. Understanding how RBC deformability and blood Hct levels impact WBC and VTC margination and behavior in blood flow has the potential to elucidate disease mechanisms and viable routes for VTC diagnostics and treatments. 

References:

1. Crowl et al. International Journal for Numerical Methods in Biomedical Engineering(2010) vol 26, 471–487.

2. Kumar, A. & Graham, M. D. Physical Review Letters (2012) vol 109.

3. Charoenphol et al. Biomaterials (2010) vol 31, 1392-1402.