The microfluidic assay used to assess adhesion of sickle RBCs to immobilized ICAM-1 under physiological flow conditions. (A) Assembled microdevice containing 3 separate microchannels, each functionalized with ICAM-1. Arrow indicates the flow direction. (B) Recombinant human ICAM-1 protein was immobilized on the microchannel surface via a cross-linker agent, GMBS, to ensure consistent protein coverage throughout the microchannel, as well as to prevent protein dissociation from the surface at high shear rates. (C) Representative image obtained following an adhesion experiment (upper panel). Enlarged images of the green boxes are shown (lower panel). Adherent sickle RBC populations possessed distinct morphologies: (i) RBC with a characteristic biconcave morphology and elongated elliptic shape, (ii) RBC with a nearly absent biconcave morphology and further impaired elliptic shape, (iii) RBC with no biconcave morphology and elongated elliptic shape, (iv) highly sickled RBC with no biconcave morphology. (D) The number of adherent RBCs was significantly greater in samples from subjects with HbSS > HbS variant > HbAA. The horizontal lines represent the means; ‘‘n’’ represents the number of blood samples tested. A total of 106 blood samples were tested from 55 subjects with HbSS SCD. The scale bars are 5 μm.