Background: Activation of complement is a major barrier in xenotransplantation. We report a detailed monitoring strategy in a 58-year-old man who received a 10-gene–edited porcine heart. Despite expression of human complement regulators CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor) in the xenoheart, early biopsies revealed significant complement deposition, prompting changes in therapeutic approach.
Methods: The initial strategy for the inhibition of complement used the C1 esterase inhibitor (C1INH) to target classical/lectin pathways. After the first endocardial biopsy performed on day 13, the endomyocardial biopsy (EMB) showed diffuse capillary C3d and C4d deposition, therapy was switched to a C5 inhibitor, eculizumab. We monitored complement activity via immunohistochemical staining (C3c, C3d, C4d, C5b-9) on biopsies, serum soluble C5b-9 (sMAC) levels via ELISA, and a bioluminescent modified Ham test (bmHam) using HEK293 cells lacking complement regulators (CD46, CD55, CD59). The bmHam assay measured serum-mediated cell lysis and included titrated mixes with normal human serum to gauge complement inhibition functional strength.
Results: EMB (POD13) revealed widespread C3c, C3d, and C4d capillary deposition and endothelial activation, suggesting early innate complement-mediated injury. After initiating eculizumab, upstream complement fragments (C3c, C3d, C4d) remained detectable in biopsies (as expected, given known tissue persistence), but capillary C5b-9 deposition was only focally evident on later biopsy (POD30) (Figure 1). Ex vivo assays indicated that switching from C1INH to eculizumab improved complement control: serum sC5b-9 levels dropped to low levels, and bmHam showed reduced complement-mediated lysis consistent with effective terminal pathway blockade (Figure 2A). In dilutional bmHam tests, patient serum obtained on eculizumab required significantly higher proportions of normal serum to reach 50% cytotoxicity indicating stronger complement blockade compared to serum on C1INH. Notably, plasma exchange (TPE) and massive transfusion events correlated with transient rises in sC5b-9 and bmHam cytotoxicity, suggesting reduced drug levels and complement “breakthrough” that were promptly identified by these assays (Figure 2B).

Conclusions: Activation of complement contributed to early porcine-to-human cardiac xenograft injury despite transgenic human complement-regulatory transgene expression. A shift from upstream (C1) to terminal (C5) inhibition aligned with marked reduction in circulating and tissue terminal complement activity. Immunohistochemical detection of C3d/C4d provided evidence of complement activation but could not discriminate ongoing injury due expected persistence of tissue complement deposits. By contrast, real-time assays (sC5b-9 and bmHam) proved valuable for monitoring functional complement activity and guiding therapeutic adjustments.