C3 coating on RBCs by flow cytometry. (A) Single-color flow cytometry. (y-axis) Cell count; (x-axis) FITC-conjugated anti-C3 polyclonal antibody (logarithm of fluorescence). Each filled histogram represents a single case, with the corresponding isotypic control (empty histogram). CAD indicates cold agglutinin disease (positive control). In the PNH patients, a discrete population of RBCs coated by C3 appears under eculizumab treatment. (B) Double-color flow cytometry. (y-axis) PE-conjugated anti-CD59 monoclonal antibody (logarithm of fluorescence); (x-axis) FITC-conjugated anti-C3 polyclonal antibody (logarithm of fluorescence). Each dot plot represents a single case. The typical bimodal pattern (CD59⁺ and CD59⁻) of untreated PNH patient becomes trimodal under eculizumab treatment for the presence of CD59⁻/C3⁺ RBCs.