Figure 1.

Complement activation in AIHA is fully CP‐mediated. (a) Sera of AIHA patients and NHS as complement source (25%, v/v) were incubated with healthy donor RBCs in the presence or absence of CP inhibitor anti‐C1q. C4 and C3 deposition was determined (black line) showing that incubation with anti‐C1q (green line) completely inhibited complement activation to background levels (HI‐NHS solid grey). (b, c) Sera of three AIHA patients (with ●, ▲ and ■ depicting individual patients) and FB‐depleted serum (red line) or FD‐depleted serum (blue line) supplemented with or without FB and FD respectively (solid blue/red versus dotted line) as complement source (25%, v/v) were incubated with healthy donor RBCs in the presence or absence of CP inhibitor anti‐C1q (green line) and C4 (a, b) or C3 (a–c) deposition was determined. C4 and C3 deposition in the absence or presence of FB or FD was equal in all situations. Anti‐C1q completely inhibited complement activation under all conditions. (d) Haemolysis of RBCs induced by AIHA patient antibodies, relative to 100% lysis by incubation with milliQ. (e) C3 deposition was measured on HAP1 CD46/CD55 KO cells, showing that in the absence of these complement regulators, the presence of FB or FD did not affect complement activation, while anti‐C1q could completely block complement activation. (a) Histograms for AIHA patient 1. (b–d) Mean with SD of three different AIHA patient sera. (e) Mean with SD of n = 3 independent experiments. Statistical significance was tested using the Friedman test with Dunn's multiple comparisons test for b, c, e and using repeated measures ANOVA with Dunnett's multiple comparisons test for d.