FACS analysis of C5 binding to PNH-RBCs loaded with C3b or C3dg. PNH-PBCs were loaded with C3b in human factor I–depleted serum under combined C5 inhibition (with eculizumab and coversin) to allow for efficient C3b opsonization without lysis. A portion of the C3b loaded cells were exposed to factor I to allow for processing of the surface-bound C3b to C3dg. (A) Correlation of C3b deposition levels with PNH subtype. Detection of expression levels of CD59 (x-axis) allows classification of PNH-RBCs in type I (normal CD59 levels), type II (reduced level), and type III erythrocytes (absence of CD59). C3b deposition (y-axis) is more pronounced for type III PNH-RBCs than for type II or type I. (B) Correlation of C5 binding with C3b surface levels. C5 binding on PNH-RBCs coincides with high C3b density (1 representative assay from 2 independent experiments is shown).