*Purpose: The successful use of hypothermic machine perfusion (HMP) as a clinical modality for graft preservation prior to kidney transplantation (KTx) has generated a demand for novel strategies aimed at improving graft viability assessment and facilitating prediction of early post-KTx graft function and survival. Circulating cell-free DNA (cfDNA) released from injured, necrotic, and apoptotic cells is an emerging biomarker of tissue injury. Here we test if cfDNA levels in the perfusate of human kidney allografts undergoing HMP is an accurate measure of preservation quality and post-transplant renal function.

*Methods: Perfusate samples of 12 kidney grafts selected for HMP at the University of Florida were collected after 5 minutes and at conclusion of HMP. Graft recipients were enrolled under an IRB approved protocol. cfDNA was quantified by real-time polymerase chain reaction using specific primers sequences for DNA of nuclear origin and correlated with both HMP parameters and post-kidney transplant clinical outcomes. The primary outcome was delayed graft function (DGF) and secondary outcomes were clinical measures of early graft function up to post-operative day (POD) 4

*Results: Kidney grafts included in this assessment had a mean 727 min (SD: 376) of static preservation on ice, a mean 419 min (SD: 155) of HMP and a mean 1153 min (SD: 358) of total cold ischemia. There were no cases of DGF. 5 min perfusate cfDNA levels correlated positively with the graft’s initial renal flow on the pump (ρ=0.863 p=0.0003) and negatively with the graft’s initial renal resistance on the pump (ρ=-0.791 p=0.011). Interestingly, grafts with higher levels of perfusate cfDNA at HMP conclusion had reduced graft function in the initial post-KTx period. Increased endpoint perfusate cfDNA concentrations correlated with increased recipient serum creatinine levels at POD4 (R²=0.452, p=0.016), decreased POD4 estimated glomerular filtration rate (R²=0.361, p=0.039) and decreased creatinine reduction ratio (R²=0.753, p=0.0005). In contrast, there was no observed correlation between clinical measures of early post-KTx graft function and endpoint renal resistance or renal flow readings for these grafts

*Conclusions: These findings provide initial evidence that quantification of cfDNA content in the perfusate of ex vivo hypothermic perfused kidney grafts can provide insight to the quality of preservation of these grafts and their early post-transplant renal function.

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