Background: Organ characteristics and quality play a critical role in the degree of ischemia/reperfusion injury (IRI) and graft function, especially in marginal organs. This study aims to determine differential molecular hallmarks in IRI between standard and extended criteria donors.

Patients and Methods: Deceased donor LT recipients (n=41) were included. Graft biopsies (n=82) were collected in pairs at pre-implantation (Pre-I) and 90 minutes post-reperfusion (Post-R). Grafts included in the Training set (n=24) were grouped as standard (SCD, n=13) and extended (ECD, n=11) criteria donors. ECD classification was defined as: donor age ≥ 60-yo, CIT ≥ 8 hrs, WIT ≥ 60 min, and non-heart beating donors. Validation set included 17 LT recipients. Total RNA was isolated and used for microRNA (miRNA) and gene expression (GE) microarrays hybridization. Probeset summaries were obtained using Robust Multiarray Analysis (RMA) algorithm. Pairwise comparisons were performed as follow: ECD vs. SCD at Pre-I, Post-R vs. Pre-I for ECD, Post-R vs. Pre-I for SCD, and Post-R vs. Pre-I. Comparisons were fit using two-sample t-test. Significant considered p-values ≤ 0.001 (GE) and ≤ 0.01 (miRNA). FDR was controlled ≤ 5%. Molecular pathway analyses were performed using Ingenuity Pathway Analysis (IPA) tool. Top significant miRNAs and genes were assessed by qPCR in the Validation set.

Results: No significant differences in clinical and laboratory (LFTs, TB, ALP, INR and LOS) parameters were observed between groups. ECD group donors were older (p=0.04), male (p=0.01), and increased BMI (p = 0.027). Significant differential GE profiles for ECD grafts (684 genes/30 miRNAs) and SCD (1011 genes/15 miRNAs) were identified at Post-R. Of those, 64 and 52 molecules were unique for each group. Molecular pathway analyses evidenced inflammatory processes in both groups. However, the level of response to the inflammatory process was different and exacerbated in the ECD group. Indeed, molecular profile in ECD graft favored mononuclear and antigen presentation cells movement and activation. Furthermore, ECD molecular profile was associated with increased synthesis and accumulation of lipids and free radicals production. Top five significant molecules were independently validated.

Conclusion: Despite inflammatory processes were observed in IRI for all liver grafts, the ECD group seems to have an abnormally exacerbated response aggravated by accumulation of lipids and free radicals.

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