Complement biosensors identify a range of “normal” complement activation in healthy control sera. (A) Overview of creation of biosensors with different susceptibilities to complement and description of the assay. (B-E) Healthy control (HC) or heat-inactivated sera incubated with Livelight WT HEK293 cells (B), CD46KO cells (C), PIGAKO cells (D), double CD46, and PIGA KO (DKO) cells (E). Relative luminescence measured every 5 minutes for 2.5 hours. Data plotted as mean ± standard deviation (SD) for each triplicate. (F) Summary of relative luminescence at 1 hour for healthy controls (WT, CD46KO, and PIGAKO, n = 19; and for DKO, n = 18). Asterisk identifies outlier by ROUT analysis (Q = 1%). P values were calculated using 1-way analysis of variance (ANOVA) for Dunnett multiple-comparisons test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001, ∗∗∗∗P < .0001. HI, heat inactivation; ns, not significant; RLU, relative luminescence units; ROUT, robust regression and outlier. Portions of figure were created with BioRender.com.