*3.5. dd-cfDNA*

Small fragments of cell-free DNA, released from graft cells into the recipient circulation due to cell death or injury, have been proposed as biomarkers of AR.

While dd-cf DNA represents on average 0.34% of total cf-DNA in plasma of stable KTx recipients, levels are increased during AR and, to a lesser extent, acute pyelonephritis and ATN.

A kinetic pilot study of dd-cfDNA after Tx showed high median level in the immediate post-Tx hours (around 20%), rapidly decreasing on the first day (around 5%) and then stabilizing below 1% [107].

In the DART (Diagnosing Active Rejection in Kidney Transplant Recipients) trial, Bloom RD et al. first reported higher dd-cfDNA levels in patients with acute (TCMR Banff > IB and ABMR) and chronic

active rejection and identified a 1% threshold to discriminate these patients from stable ones. This test was characterized by elevated NPP (84%) and a lower PPP (61%), suggesting that <1% percentage of dd-cfDNA could be used to rule out rejection, especially ABMR. Coexistence of DSA increased PPP to 85%. Furthermore, dd-cfDNA levels not only increased before changes in serum creatinine but also decreased after rejection treatment, suggesting that longitudinal monitoring of this biomarker could be useful after a rejection episode, possibly limiting need for surveillance biopsies [67].

In a subsequent work, Huang E et al. [68] demonstrated that a lower dd-cfDNA threshold of 0.74% could reliably identify ABMR—but not TCMR—in a group of immunologically high-risk patients undergoing indication biopsies, increasing NPP to 100%.

However, other authors suggested comparable performance of dd-cfDNA in diagnosing ABMR and TCMR, using a different quantification methodology [69].

Absolute quantification of dd-cfDNA (copies/mL) showed superior performance in discriminating BPAR as compared to dd-cfDNA percentage and also seemed to identify a subset of patients with inadequate Tacrolimus levels and subclinical immunological damage in a prospective observational study [70].

Of interest, dd-cfDNA diagnostic capacity for ABMR appears to improve when applied to DSA-positive recipients, suggesting a preferential employment in monitoring highly sensitized patients [71].

Determination of dd-cfDNA can be unreliable in case of recent (within 1 month) whole blood transfusion and falsely positive within 24 h of a renal biopsy; it should also not be employed to monitor a second KTx as release from previous graft could alter its levels. Falsely positive results can also occur in the case of ATN and acute pyelonephritis and type of donor also affects levels (higher levels in cadaveric vs. LD), probably reflecting difference in degree of initial ischemic damage and inflammation [72].

Despite these issues, dd-cfDNA remains a promising biomarker and it has been proposed as a surrogate diagnostic ABMR criterion in DSA-negative forms [108].

Furthermore, recent studies suggest that dd-cfDNA determination could also have a broader meaning beyond AR diagnosis, reflecting graft injury and consequently exerting a negative impact on several long-term outcomes [109]. Of interest, a multicentric study on patients with initial TCMR (TCMR 1A and borderline lesions) showed that dd-cfDNA levels above 0.5% were effective in stratifying risk of eGFR decline, de novo DSA development and further AR episodes [110]. Consistently, emerging evidence indicates that levels of dd-cfDNA increase before onset of de novo DSA (both HLA-DSA and non-HLA DSA) and eGFR decline [73], suggesting that dd-cf DNA itself is immunogenic and can trigger subclinical inflammation, initiating an immune response [75].

Urinary levels of cell-free mitochondrial DNA during early post-transplant phase have also been reported to correlate with AR, DGF and short-term renal function [76].
