Figure 2.
Example CM-TMA tracings and summary of relative luminescence and blocking. (A,D) CM02 sera on CD46KO cells (A) or PIGAKO cells (D) in GVB++ with or without heat inactivation (HI), eculizumab (ecu; 50 μg/mL), sutimlimab (sut; 30 μg/mL), or ACH-5548 (FDi). (B,E) Summary of relative 1-hour luminescence of healthy controls (HC), CM-TMA (acute or remission) with or without the addition of ecu, sut, ACH-4471/ACH-5548 (FDi; 1 μM), iptacopan (FBi; 1.5 μM), or “triple AP blockade” with FDi (1 μM), FBi (1.5 μM), and compstatin (18 μM) on CD46KO (B) or PIGAKO (E) (PIGAKO: HC, n = 18; acute TTP, n = 6; acute CM-TMA, n = 5; remission, n = 14; CM-TMA + ecu, n = 12; CM-TMA + sut, n = 11; CM-TMA + FDi, n = 11; CD46KO: HC, n = 18; acute TTP, n = 6; acute CM-TMA, n = 5; remission CM-TMA, n = 13; CM-TMA + ecu, n = 8; CM-TMA + sut, n = 8; CM-TMA + FDi, n = 7; CM-TMA FBi, n = 3; CM-TMA triple blockade, n = 3). Graphed as mean ± SD. Yellow fill indicates sample with pathogenic variant or variant of uncertain significance (VUS) in complement regulatory protein. Dashed line represents the 15th percentile of healthy control. (C,F) Relative 1-hour luminescence recovery after addition of inhibitor calculated at 1 hour as follows: (luminescence inhibitor-treated serum − luminescence untreated serum)/(luminescence heat-inactivated serum − luminescence untreated serum); (PIGAKO, n = 10 for each condition; CD46KO: CM-TMA + ecu, n = 12; CM-TMA + sut, n = 9; CM-TMA + FDi, n = 8; CM-TMA + FBi, n = 3; triple blockade, n = 3). (B,C,E-F) P values were calculated using 1-way ANOVA for Dunnett multiple-comparisons test. (G-H) AP only buffer inhibits complement activity. CM12 sera on PIGAKO in GVB++ (G) or MgEGTA (AP only buffer with 13 mM Mg2+) (H) with or without inhibitors. (I) Example tracing of acute CM-TMA CM03 (FHAA) on CD46KO with or without inhibitors. (J) Example tracing of remission CM-TMA no mutation CM01 on CD46KO with addition of iptacopan (FBi; 1.5 μM). (K) Example tracing of CM13 on CD46KO with addition of “triple AP blockade” including with FDi (1 μM), FBi (1.5 μM), and compstatin (18 μM). (L) Example tracing of C3 glomerulopathy (C3G) with VUS in CD46 on CD46KO. Activity persists despite C3 being below limit of detection at time sample was collected (<15 mg/dL). Data plotted as mean ± SD for each triplicate. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant; RLU, relative luminescence units.

Example CM-TMA tracings and summary of relative luminescence and blocking. (A,D) CM02 sera on CD46KO cells (A) or PIGAKO cells (D) in GVB++ with or without heat inactivation (HI), eculizumab (ecu; 50 μg/mL), sutimlimab (sut; 30 μg/mL), or ACH-5548 (FDi). (B,E) Summary of relative 1-hour luminescence of healthy controls (HC), CM-TMA (acute or remission) with or without the addition of ecu, sut, ACH-4471/ACH-5548 (FDi; 1 μM), iptacopan (FBi; 1.5 μM), or “triple AP blockade” with FDi (1 μM), FBi (1.5 μM), and compstatin (18 μM) on CD46KO (B) or PIGAKO (E) (PIGAKO: HC, n = 18; acute TTP, n = 6; acute CM-TMA, n = 5; remission, n = 14; CM-TMA + ecu, n = 12; CM-TMA + sut, n = 11; CM-TMA + FDi, n = 11; CD46KO: HC, n = 18; acute TTP, n = 6; acute CM-TMA, n = 5; remission CM-TMA, n = 13; CM-TMA + ecu, n = 8; CM-TMA + sut, n = 8; CM-TMA + FDi, n = 7; CM-TMA FBi, n = 3; CM-TMA triple blockade, n = 3). Graphed as mean ± SD. Yellow fill indicates sample with pathogenic variant or variant of uncertain significance (VUS) in complement regulatory protein. Dashed line represents the 15th percentile of healthy control. (C,F) Relative 1-hour luminescence recovery after addition of inhibitor calculated at 1 hour as follows: (luminescence inhibitor-treated serum − luminescence untreated serum)/(luminescence heat-inactivated serum − luminescence untreated serum); (PIGAKO, n = 10 for each condition; CD46KO: CM-TMA + ecu, n = 12; CM-TMA + sut, n = 9; CM-TMA + FDi, n = 8; CM-TMA + FBi, n = 3; triple blockade, n = 3). (B,C,E-F) P values were calculated using 1-way ANOVA for Dunnett multiple-comparisons test. (G-H) AP only buffer inhibits complement activity. CM12 sera on PIGAKO in GVB++ (G) or MgEGTA (AP only buffer with 13 mM Mg2+) (H) with or without inhibitors. (I) Example tracing of acute CM-TMA CM03 (FHAA) on CD46KO with or without inhibitors. (J) Example tracing of remission CM-TMA no mutation CM01 on CD46KO with addition of iptacopan (FBi; 1.5 μM). (K) Example tracing of CM13 on CD46KO with addition of “triple AP blockade” including with FDi (1 μM), FBi (1.5 μM), and compstatin (18 μM). (L) Example tracing of C3 glomerulopathy (C3G) with VUS in CD46 on CD46KO. Activity persists despite C3 being below limit of detection at time sample was collected (<15 mg/dL). Data plotted as mean ± SD for each triplicate. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, not significant; RLU, relative luminescence units.

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