Heterogeneous suspensions containing pdRBCs and sRBCs exhibit margination toward vessel walls leading to increased endothelial dysfunction in large venule-mimicking microfluidic systems. (A) Macroscopic view of a 50 × 100 μm straight-channel microfluidic device designed to mimic a large venule. (B) Cartoon depiction of our microfluidic experimental setup and corresponding video snapshot of a superposition of brightfield and fluorescence microscopy images of an RBC suspension comprising 5% pharmacologically dehydrated, less-deformable RBCs (stained with octadecyl rhodamine B chloride [R18] stain) and 95% hRBCs flowing through a straight microfluidic channel with a diameter of 100 μm at a constant flow rate. R18-stained RBCs traveling close to the channel wall as well as more centrally in the channel are circled by a dashed yellow line and solid line, respectively. (C) Graphical representation of the experiment shown in panel B, comparing an RBC suspension composed of 100% hRBCs (5% RBCs stained) with an RBC suspension with a majority hRBC population (95%) with a minority less-deformable and stained RBCs (5%). Software analysis tracking of the stained RBCs was performed and showed that, compared with hRBCs, less-deformable RBCs were more likely to marginate and travel in close proximity to vessel walls. (D) Endothelial VCAM-1 (green) and E-selectin (red) expression after 4-hour perfusion of healthy control RBCs vs RBC suspensions containing varying amounts (5%, 10%, and 100%) of sRBCs. All RBC suspensions were diluted in media to a hematocrit of 25% and perfused at a constant venular shear rate. Graphical representation of mean normalized fluorescent intensity for VCAM-1 (left) and E-selectin (right), both markers of inflammation, in ECs exposed to hRBCs (red) and RBC suspensions with varying amounts of sRBCs in the straight-channel microfluidic is also shown. A total of 5 separate experiments were analyzed. VCAM-1 and E-selectin expression increased as the amount of sRBCs increased (VCAM-1, 1.35 ± 0.12 SEM [P < .05]; E-selectin, 1.29 ± 0.10 SEM [P < .05]) normalized fluorescent intensity in the 100% sRBC-exposed ECs. Because sRBCs do not represent only ISCs (ie, the least-deformable RBCs), the 5% and 10% sickle suspensions likely did not contain enough of the least-deformable RBCs to lead to increased margination compared with the 100% sRBC suspension. (E) Endothelial VCAM-1 and E-selectin expression after 4-hour perfusion of healthy control RBCs vs RBC suspensions containing varying amounts (5%, 10%, and 100%) of nystatin-treated RBCs, with mean normalized fluorescent intensity for VCAM-1 (left) and E-selectin (right) in ECs exposed to hRBCs (red) and RBC suspensions with varying amounts of nystatin-treated RBCs in the straight-channel microfluidic. A total of 5 separate experiments were analyzed. VCAM-1 and E-selectin expression in heterogeneous suspensions with a minority of less-deformable cells at 5% or 10% of the total suspension showed greater expression than the 100% low-deformability suspension (VCAM-1, 1.7 ± 0.17 SEM [P < .05]; E-selectin, 2.91 ± 1.56 SEM [P < .05] normalized fluorescent intensity highest in 10% nystatin-treated RBC-exposed ECs), supporting our hypothesis that when the less-deformable cells are a minority subpopulation, there is increased margination and RBC-EC interactions and subsequent dysfunction. Statistical analyses using a Mann-Whitney U test; ∗P ≤ .05. Error bars represent SEM.