Introduction: C4d staining in peritubular capillaries has been part of antibody-mediated rejection (ABMR) definition in the Banff Classification since 2003. However, despite relatively high specificity, c4d staining shows limited sensitivity, and the clinical significance of C4d+ biopsies without other histological evidence of rejection (WER) is unknown. We aimed to assess the clinical significance of c4d+ biopsies WER on kidney biopsies after ABO-compatible transplantation in pediatric recipients

Methods: We retrospectively analyzed all patients under 18 years of age with a diagnosis of c4d+ WER on kidney biopsies performed between 2011 and 2020 in the two pediatric transplant centers in Paris, France. All biopsies were reviewed by a single expert pathologist to confirm the diagnosis of positive C4d immunohistochemical staining and to ensure the absence of ABMR (Banff g0 cpt0 v0 cg0) or TCMR (≤ i2 t2). Patients’ characteristics and graft survival without ABMR were compared with a cohort of C4d-negative patients matched on recipients’ demographics, year and center of transplantation, biopsy indication and time to transplantation. Means and proportions were compared using a Mann-Whitney or the Fisher exact test. Survival was compared using Kaplan Meier curves and log rank test.
Results: 6% of biopsies performed during the study period had C4d+ WER. The 40 children with C4d+ WER were compared with 40 matched C4d- controls (median age 12 and 11,6 years). The majority (65%) were protocol biopsies performed within the first year after transplant (median 6,9 months, IQR 2,5 – 15,2). There was no significant difference between the groups regarding recipient, donor, or transplant characteristics. However, C4d+ patients had significantly more class I and II HLA mismatches at transplantation (mean 4,3 versus 3,6, p = 0,026) and positive DSA on the day of biopsy (40% versus 17,5% of patients, p=0,047). After a median follow-up of 4 years after the biopsy of interest, 4/40 C4d+ patients developed an ABMR within the first year (median 10,4 months, IQR 6,2 – 14,0). All these patients had positive DSA at the time of biopsy. However, among the patients who had a follow-up biopsy (n=32), we noted a negativation of C4d staining in 78%, including 20/32 patients (62%) in the absence of specific treatment. Survival rate without ABMR did not differ between C4d+ and C4d- patients (Fig A). However, C4d+ DSA+ patients developed significantly more ABMR within the first-year post biopsy compared with C4d+DSA- and C4d- patients (Fig B). Interestingly, patient characteristics and outcomes of C4d+DSA- patients did not differ from C4d- patients.
Conclusion: C4d+ staining WER and without DSA at the time of biopsy do not represent an increased risk of ABMR compared to C4d- patients. Screening for non-HLA antibodies and molecular analysis of the biopsies may help to assess the significance and clinical implication of C4d+ staining WER in pediatric kidney transplant recipients.