Donor-derived cell-free DNA and its utility in kidney transplantation: A myth or a reality

Table 1 Summaries of studies utilizing donor-derived cell-free DNA assay in the diagnosis of rejections

Ref.	Journal/year	Objective	Area under the curve	Threshold dd-cfDNA	Sensitivity and specificity/predictive value	Findings
Bloom et al[13]	J Am Soc Nephrol/2017	To investigate dd-cfDNA is a marker of rejection	0.74 for TCMR and ABMR	1%	PPV 61%; NPV 84%	Levels above 1% indicate a probability of active rejection of TCMR (> BANFF 1 B) or ABMR
Sigdel et al[14]	Clin Med/2018	To study single nucleotide polymorphism (SNP) multiplexed PCR to measure dd-cfDNA in various types for the detection of allograft rejection/injury	0.87	1%	Sensitivity 89%; specificity 73%; PPV 52%; NPV 95%	SNP-based dd-cfDNA assay detected allograft rejection with good performance
Huang et al[15]	Am J Transplant/2019	To investigate dd-cfDNA is a marker of rejection	0.42 for TCMR, 0.82 for ABMR	0.74%	Sensitivity 100% for ABMR; Specificity 71.8% for ABMR; PPV 68.6% for ABMR; NPV 100% ABMR	dd-cfDNA could not discriminate TCMR from no rejection -dd-cfDNA strongly discriminates TCMR from no rejection
Whitlam et al[16]	Am J Transplant/2019	To evaluate the validity of dd-cfDNA, total cell-free DNA, and graft fraction to correlate with individual Banff scores	0.89%	0.75%	Sensitivity 85%; PPV 48%; NPV 95%	dd-cfDNA was more associated with AMR -Failed to diagnose borderline or type 1A TCMR
Wijtvliet et al[17]	Transpl Int/2020	Metanalysis on dd-cfDNA to predict rejection				dd-cfDNA is a useful marker for ABMR but not for TCMR
Dauber et al[18]	Transpl Int./2020	To use INDEL PCR for dd-cfDNA for the detection of TCMR	0.84 to discriminate TCMR from no rejection	2.7%	Sensitivity 88%; Specificity 81%	INDEL PCR for dd-cfDNA could discriminate between TCMR and no rejection at a median level of 5.24%
Zhang et al[19]	Front Immunol /2020	To evaluate the diagnostic performance of dd-cfDNA in discriminating antibody-mediated rejection (ABMR) or de novo donor-specific antibodies (DSA) without histological lesions in kidney allograft recipients	0.90	1%	Sensitivity 89%; sensitivity 74%; PPV 76%; NPV 88%	dd-cfDNA correlates with AMR and may pick up cases of ABMR with stable graft function, and can help in early diagnosis
Benning et al[20]	Transpl Int/2023	To investigate dd-cf DNA in predicting rejections in patients with graft biopsy and see response to treatment	AUC 0.72 for any rejection including borderline vs no rejection		Median 2% for ABMR; median 0.92% for TCMR; median 0.42% for borderline rejection	dd-cfDNA distinguishes between active rejection and no rejection. dd-cfDNA level decreased due to antirejection therapy
Xing et al[21]	Biomol Biomed/2024	Metanalysis on dd-cfDNA to predict rejection	AUC for TCMR 0.80, AUC for ABMR 0.87		Sensitivity for TCMR 59% and specificity 83%; sensitivity for ABMR 81% and specificity 80%	Diagnostic ability for TCMR is limited, but it is a useful marker to detect ABMR
Aubert et al[22]	Nat Med/2024	Observational multicentric study of 1134 patients	AUC from 0.77-0.82	2.85% ± 0.68% for mixed rejection; 2.03% ± 1.13% for acute TCMR; 1.15% ± 0.15% for active AMR; 1.09% ± 0.15% for chronic active AMR		dd-cfDNA strongly correlated with ABMR, TCMR, and mixed rejection
Akifova et al[23]	Nephrol Dial Transplant/2024	A randomized prospective study in recipients with donor-specific antibody (DSA) and high dd-cfDNA and DSA with clinical indication of biopsy was compared		> 50 cp/mL	PPV 77%; NPV 85%	Biopsy in dd-cfDNA was performed earlier, leading to early management

dd-cfDNA: Donor-derived cell-free DNA; TCMR: T cell–mediated rejection; ABMR/AMR: Antibody-mediated rejection; DSA: Donor-specific antibody; SNP: Single nucleotide polymorphism; INDEL: Insertion–deletion; PCR: Polymerase chain reaction; AUC: Area under the curve; PPV: Positive predictive value; NPV: Negative predictive value; Banff: Standardized histopathological classification for kidney allograft pathology; IFTA: Interstitial fibrosis and tubular atrophy; DGF: Delayed graft function.

Table 2 Summaries of studies of Allosure, Prospera, Viracor TRAC, and their diagnostic abilities

Ref.	Journal/year	Assay used	SNPs	Limit of quantification	Limit of detection	Sensitivity	Specificity	Area under curve	Coefficient of variation
Grskovic et al[48]	J Mol Diagn/2016	Allosure	266	0.2%	0.16%	0.83	0.84		7.7% (dd-cfDNA < 2%). 4.5% (dd-cfDNA ≥ 2%)
Bloom et al[13]	J Am Soc Nephrol/2017	Allosure	266			0.59	0.85	0.74
Jordan et al[49]	Transplant Direct/2018	Allosure	266	0.2%-16%				0.86
Sigdel et al[14]	J Clin Med/2018	Prospera	13392			0.887	0.726	0.87 (87%)
Altuğ et al[50]	Transplantation/2019	Prospera	13962	0.15% (related donors); 0.23% (unrelated donor)	0.15% (related donors); 0.23% (unrelated donor)				4.29%
Melancon et al[51]	Kidney360/2020	Allosure vs Prospera	266 vs 13392			0.45 vs 0.55	0.85 vs 0.69	0.73 (73%) vs 75 0.75 (75%)
	Viracor TRAC	Quantitative PCR	0.5%-60%		0.579		0.853

dd-cfDNA: Donor-derived cell-free DNA; SNP: Single nucleotide polymorphism.