Fig. 4

Effect of complement activation by mild acidification on PNH red cells from patients on eculizumab. a C3 binding on PNH red cells from a representative PNH patient on eculizumab after complement activation in vitro by mild acidification in presence of eculizumab. This experiment shows the C3 binding (C3+) on PNH (CD59-negative) red cells before (left panel) and after 6 h (right panel) from complement activation in vitro. The number in the upper right angle of each diagram indicates the time in hours (h); 0 h indicates the sample analyzed before complement activation in vitro. b Kinetics of C3 binding on PNH red cells from a series of PNH patients on eculizumab after complement activation in vitro by mild acidification in presence of eculizumab. The line diagram shows the average (+SD) proportion of C3-negative PNH red cells that became bound with C3 in a series of eculizumab-treated patients at different time points (from 1 to 24 h) since complement activation in their own sera (continuous line without MgCl₂; dotted line with 1.25 mM MgCl₂). In this 7 patients, the percentage PNH red cells that in vivo were already bound with C3 was 30.3 ± 24.5%. n: number of different patients studied at the indicated time points. c Effect of in vitro complement activation on C3− PNH red cells from patients on eculizumab. RBCs without C3 binding have been sorted by FACS from a patient on eculizumab and subjected to complement activation in the presence of eculizumab. Analysis of RBC with anti-CD59 and anti-C3d: just after sample drawing (left panel); after FACS sorting of C3− RBC (middle panel); sorted C3− RBC after 2 h from complement activation in presence of eculizumab (right panel). HCl: serum acidification. ECU: eculizumab