CM-TMA sera has increased IgM binding to HEK293 cell surfaces and can presensitize cells even after HI of serum. Heat-inactivated sera was used for all cytometry and presensitization experiments to eliminate preloading of cells with C3 or C4 fragments. (A) WT cells were incubated for 45 minutes at 37°C with either heat-inactivated HC or CM-TMA sera (20% in GVB⁺⁺), washed, and evaluated for deposition of IgG (HC, n = 9; CM-TMA n = 17) or IgM (HC, n = 7; CM-TMA, n = 15) by flow cytometry. Acute CM-TMA samples (n = 6) indicated by red dots. Relative MFI calculated as ratio of sample MFI compared with the average of 4 healthy controls. P values were calculated using unpaired, 2-tailed t test, with Welch correction. (B-C) Example histograms of IgG (left) or IgM (right) staining from CM21 (B) and CM04 (C) on WT cells with (red) or without (blue) pretreatment of serum with DTT (3 mM pretreatment). HC stained IgG or IgM stained cells included as baseline reference population (gray). (D) Bioluminescent mHam tracing of HC1 on DKO. (H) Bioluminescent mHam tracing of HC2 on PIGAKO. (E-G,I-K) Specially selected low activity healthy control sera mix was used to facilitate complement activity in the bioluminescent mHam after either DKO (E-G) or PIGAKO (I-K) cells were presensitized (20% heat-inactivated sera treatment for 45 minutes in Dulbecco’s modified Eagle medium), washed, and resuspended in GVB⁺⁺ then run in the bioluminescent mHam. Baseline activity of the healthy control mix sera shown in each tracing as dark blue (HC sera mix) and dark red (HI HC sera mix). Pretreatment sera included HC1 (E), CM21 (F), CM04 (G), HC2 (I), CM07 (J), and CM06 (K). (D-K) Tracings plotted as mean ± SD for each triplicate. ns, not significant; RLU, relative luminescence units.