Clearance of C3d Immunostaining Occurs More Often and Earlier Than Clearance of C4d after Antibody Mediated Rejection: An Earlier Marker for Resolving Rejection?

The 2013 ISHLT Working Formulation for antibody mediated rejection (AMR) includes C3d and C4d staining as part of the immunopathology diagnostic criteria. Though C3d and C4d are both incidental complement split products, they differ in important ways. For example, per every unit of C1 initiating the complement cascade, 8.6 times more C3 is cleaved than C4. While it is stoichiometrically more abundant, the kinetics of its clearance once covalently bound to endothelium is not well studied. We sought to investigate anecdotal observations suggesting that immunopathologic staining for C3d may not persist as long as C4d after an AMR episode. The UTAH Cardiac Transplant program’s heart biopsy database was queried for biopsies with both C3d and C4d frozen section immunofluorescence staining. The data were analyzed using a model that used each biopsy as a discrete event and then tracked subsequent biopsies within an 8 week time window. The various transition states were tabulated. The transition times from C3d & C4d positive to clearance of C3d and C4d were also calculated and compared. 6003 biopsies from 650 patients met the inclusion criteria. For 290 C3d and C4d positive biopsies, the distribution of subsequent biopsies was as follows: 163 C4d-/C3d-, 14 C4d-/C3d+, 39 C4d+/C3d-, and 74 C4d+/C3d+. The mean time to negative C3d staining was 127 days and the mean time to negative C4d staining was 146 days. Though the number of C4d and C3d positive biopsies showing clearance of C3d or C4d within 8 weeks was comparatively small, clearance of C3d was more frequent occurrence than C4d clearance. On average this happened earlier than for clearance of C4d. Our observations indicate that C3d clearance on endomyocardial biopsies could serve as a useful marker of AMR resolution.