Phagocytosis of C3dg-coated PNH erythrocytes by activated human monocytes. (A) Visualization of intracellular erythrocytes by fluorescent microscopy. The processes of phagocytosis and immunostaining are described in Methods. PNH erythrocytes were identified by positive staining with anti-C3d and anti-glycophorin antibodies. (B) Phagocytosis quantification. The results represent the average of 3 independent experiments, and the error bars represent the standard deviation. In each phagocytosis experiment, activated human monocytes were incubated with erythrocytes from a healthy donor (Ctrl) or PNH patient E (panel C). To test whether the phagocytosis is accomplished via CR3, a CR3-specific blocking antibody was preincubated with activated monocytes before adding the PNH erythrocytes, and a blocking anti-CR2 mAb was used as a nonspecific control (n = 3; *P < .05, 1-way analysis of variance). (C-D) Correlation between phagocytosis efficiency by activated human monocytes and C3dg coating of PNH erythrocytes. Phagocytosis experiments were performed with erythrocytes from 5 different PNH patients. The data in C represent the average of 3 independent experiments with error bars indicating the standard deviation. (D) Correlation between the fraction of C3dg-positive cells (supplemental Figure 4) and erythrophagocytosis (C), with error bars indicating the standard deviation (Pearson r = 0.99; P = .002).