Quantification of Graft-Derived Cell-Free DNA during Normothermic Machine Perfusion in Liver Transplantation Is Feasible and May Be a Useful Future Tool for Assessing Organ Health

Introduction: Ex-vivo normothermic machine perfusion (NMP) has demonstrated utility in achieving viability for transplantation in previously discarded liver grafts. The analysis of graft-derived cell-free DNA (gdcfDNA) is an emerging, precision tool for monitoring graft health following liver transplantation. This study sought to investigate the feasibility of measuring gdcfDNA during NMP for liver transplantation. Method: Serial perfusate and bile samples were collected during NMP for 10 liver grafts, at 15 mins post-machine reperfusion and then at two-hourly intervals until NMP completion. Samples underwent two-step high speed centrifugation to remove cellular debris. Following DNA extraction, a droplet digital PCR (ddPCR) assay was used to detect and quantify mitochondria-derived cfDNA from the graft at each time point. Results: gdcfDNA was successfully quantified in all bile and perfusate samples (n=200). Mitochondria-derived cfDNA was highly abundant; the mean concentration was more than 200x greater in bile than perfusate samples (e.g. mean concentration in bile 135 mins: 433,702,500 copies/ml vs perfusate: 1,947,600 copies/ml). There was a trend of declining gdcfDNA levels in the bile samples over time post-machine reperfusion (mean concentration 135 mins: 433,702,500 copies/ml vs 375 mins: 113,348,992 copies/ml) [see figure]. Conclusions: This is the first reported study to demonstrate that serial assessment and quantification of gdcfDNA during NMP is feasible. Graft viability during NMP is currently assessed using a combination of subjective measures and conventional biochemical tests. The measurement of gdcfDNA could form a future real-time, objective tool for assessing graft health during NMP; this will need to be evaluated in further larger studies.