aWrb scFv and hTM-scFv increase RBC rigidity, whereas aRh17 scFv and hTM-scFv show no changes compared with naive RBCs. Ektacytometry was performed on 5% Hct RBC suspensions incubated with scFv or hTM-scFv at the indicated concentrations. Elongation index (as calculated automatically by the instrument) was read as a function of shear stress, and nonlinear regression was used to calculate the shear stress required for half-maximal deformation and the EImax. Representative curves of at least 3 independent experiments with different donors. (A) hTM-scFv fusions and (C) scFv antibodies targeted to band 3/GPA (aWrb blue dotted lines) demonstrated a rightward shift in the ektacytometry curves compared with naive (solid line) whereas aRh17 fusions and scFv (red dashed lines) showed no change from naive (scFvs and fusion proteins added at 1000 nM). (B,D) The shift in deformability was quantified as the SS1/2, which showed dose-dependent increases in response to band 3/GPA-targeted ligands and not RhCE ligands. In panels B and D, mean ± SD is shown; n = 3-5 per condition (*P < .05 compared with naive, 1-way ANOVA with Holm-Sidak correction for multiple comparisons). hRBC, human RBC.
