Residual hemolysis of antibody-sensitized PNH erythrocytes in presence of eculizumab. (A) Residual TP activity under eculizumab after CP activation detected through hemolysis of PNH-RBCs. Three different alloantibodies were each incubated with erythrocytes from PNH patients (all of ABO blood group 0, positive for the antigens Jk^b, Lan, and PP1P^k), washed and exposed to standardized NHS, standardized FB-dpl serum, or serum mixed with eculizumab. The final serum content was 50%, and the final eculizumab concentration was 1.2 µM. Erythrocytes from PNH patient D were used. (Results from 1 out of 3 independent assays are shown; for the 2 other assays with erythrocytes from PNH patients E and F, see supplemental Figure 6A-B). (B) Levels of hemolysis and residual hemolysis under eculizumab correlate with the concentration of anti-PP1P^k antibody used for sensitization. PNH-RBCs were incubated with anti-PP1P^k alloantibody at different titers, washed, and then exposed to NHS or NHS supplemented with eculizumab. The final serum content was 50%. Erythrocytes from PNH patient F were used. (Results from 1 out of 3 independent assays are shown; for the 2 other assays with erythrocytes from PNH patients G and F with alloantibody anti-Jk^a and anti-Lan, see supplemental Figure 7). (C) Effect of increasing eculizumab concentrations on hemolysis of PNH-RBCs sensitized with different titers of alloantibody. Reactions were performed as in panel B. Anti-PP1P^k alloantibody and erythrocytes from PNH patient F were used. (Results from 1 out of 3 independent assays are shown; for the 2 other assays with erythrocytes from PNH patients C and F with alloantibody anti-Jr^a, see supplemental Figure 8). (D) Effect of combined complement inhibition by eculizumab and the alternative pathway inhibitor mini-FH (which specifically accelerates the natural decay of alternative pathway convertases but is a cofactor for factor I–mediated cleavage of all, classical/lectin and alternative pathway produced C3b molecules). Assay was performed as in panel B, with PNH-RBCs of patient F and anti-PP1P^k alloantibody. (Results from 1 out of 3 independent assays are shown; for other assays with erythrocytes from PNH patients E and F and anti-Jk^b alloantibody, see supplemental Figure 9). Ecu, eculizumab.