Cell-Free Dna As A Solid-Organ Transplant Biomarker: Technologies and Approaches

Figures & data

Table 1. Detection cell-free DNA in solid-organ transplant recipients.

Approaches for detecting cell-free DNA in solid-organ transplant recipients
Approach	Study	Year	Organ(s)	Recipients (n)	Targets	Technology platform	Blood collection tubes	cf-DNA isolation method	Ref.
Detecting total cf-DNA	Garcia Moreira et al.	2009	Kidney	100	HBB, TSPY1 genes	qPCR	EDTA	QIAamp DNA Blood Mini Kit	[48]
Detecting Y chromosome	Lui et al.	2003	Heart, kidney, liver	31	HBB, SRY genes	qPCR	EDTA	QIAamp DNA Blood Kit	[60]
	Macher et al.	2014	Liver	10	HBB, SRY genes	qPCR	x	Magna Pure Compact Nucleic Acid Isolation Kit I	[61]
Detecting HLA types	Zou et al.	2017	Lung	18	8 HLA-DR alleles	ddPCR	x	QIAamp Circulating Nucleic Acid Kit	[62]
	Gadi et al.	2006	Pancreas-kidney	42	HLA specific targets	qPCR	x	DNA Mini Kit	[63]
Multitarget NGS assays	Snyder et al.	2011	Heart	44	SNPs	NGS	x	Nucleospin Plasma F kit	[59]
	De Vlaminck et al.	2015	Lung	51	SNPs	NGS	x	QIAamp Circulating Nucleic Acid Kit	[42]
	De Vlaminck et al.	2014	Heart	65	SNPs	NGS	x	QIAamp Circulating Nucleic Acid Kit	[41]
	Gielis et al.	2020	Kidney	107	1027 SNPs	NGS	Streck	QIAamp Circulating Nucleic Acid Kit	[79]
	Zhang et al.	2020	Kidney	37	56,049 SNPs	NGS	x	QIAamp Circulating Nucleic Acid Kit	[80]
	Zhao et al.	2021	Liver	49	6200 SNPs	NGS	Streck	QIAamp Circulating Nucleic Acid Kit	[43]
Non-NGS multitarget assays	Hidestrand et al.	2014	Heart	32	94 SNPs	qPCR	x	QIAamp Circulating Nucleic Acid Kit	[66]
	Ragalie et al.	2018	Heart	88	94 SNPs	qPCR	x	QIAamp Circulating Nucleic Acid Kit	[82]
	Richmond et al.	2020	Heart	174	94 SNPs	qPCR	x	ReliaPrep HT Circulating Nucleic Acid Kit	[83]
	North et al.	2020	Heart	76	94 SNPs	qPCR	Streck/EDTA	Proprietary extraction protocol	[33]
	Dauber et al.	2020	Kidney	29	34 INDELs	qPCR	EDTA	QIAamp Circulating Nucleic Acid Kit	[47]
	Beck et al.	2013	Heart, kidney, liver	34	41 SNPs	ddPCR	Streck/EDTA	High Pure viral nucleic acid extraction kit	[45]
	Schutz et al.	2017	Liver	107	40 SNPs	ddPCR	Streck	High Pure viral nucleic acid extraction kit	[84]
	Knuttgen et al.	2021	Heart	87	40 SNPs	ddPCR	Streck	High Pure viral nucleic acid extraction kit	[86]
Absolute amount of DD-cfDNA	Whitlam et al.	2019	Kidney	55	38 genetic variants	ddPCR	Streck	QIAamp Circulating Nucleic Acid Kit	[89]
	Oellerich et al.	2019	Kidney	189	41 SNPs	ddPCR	EDTA	High Pure viral nucleic acid extraction LV kit	[90]

cf-DNA: Cell-free DNA; DNA: Deoxyribonucleic acid; ddPCR: Droplet digital polymerase chain reaction; DD-cfDNA: Donor-derived cell-free DNA; EDTA: Ethylenediaminetetraacetic acid; HLA: Human Leukocyte antigen; INDELs: Insertion and deletion mutations; NGS: Next-generation sequencing; qPCR: Quantitative polymerase chain reaction; SNPs: Single-nucleotide polymorphisms; Streck: Streck Cell-Free DNA BCT tubes (Streck, NE, USA).

Table 2. Commercial testing for cell-free DNA in solid-organ transplant recipients.

Commercial provider	Study	Year	Organ(s)	Recipients (n)	Targets	Technology platform	Blood collection tubes	cf-DNA isolation method	Ref.
CareDx	Bloom et al.	2017	Kidney	102	266 SNPs	NGS	Streck	QIAamp Circulating Nucleic Acid Kit	[24]
	Huang et al.	2019	Kidney	63	266 SNPs	NGS	Streck	QIAamp Circulating Nucleic Acid Kit	[68]
	Khush et al.	2019	Heart	740	266 SNPs	NGS	Streck	QIAamp Circulating Nucleic Acid Kit	[69]
	Khush et al.	2021	Lung	38	266 SNPs	NGS	Streck	QIAamp Circulating Nucleic Acid Kit	[70]
	Puliyanda et al.	2020	Kidney	67	266 SNPs	NGS	Streck	QIAamp Circulating Nucleic Acid Kit	[71]
	Sayah et al.	2020	Lung	69	266 SNPs	NGS	Streck	QIAamp Circulating Nucleic Acid Kit	[72]
	Levine et al.	2020	Lung	48	266 SNPs	NGS	Streck	QIAamp Circulating Nucleic Acid Kit	[73]
Natera	Sigdel et al.	2018	Kidney	178	13,392 SNPs	NGS	x	QIAamp Circulating Nucleic Acid Kit	[25]
	Altug et al.	2019	Kidney	6	13,392 SNPs	NGS	Streck	In-house extraction protocol/QIAamp Circulating Nucleic Acid Kit	[75]

cf-DNA isolation kit manufacturers DNA mini kit: QIAamp Circulating Nucleic acid kit, QIAamp DNA Blood kit, QIAamp DNA Blood mini kit, (Qiagen, MD, USA); High pure viral nucleic acid (LV) extraction KIT, Manga Pure Compact Nucleic acid Isolation Kit I, (Roche, Basel, Switzerland); Nucleospin Plasma F kit, (E&K Scientific Products, CA, USA) and ReliaPrep HT Circulating Nucleic Acid Kit, (Promega, WI, USA).

Figure 1. Articles related to cell-free DNA and solid-organ transplantation identified in PubMed from 2010–2020.

PubMed was searched using the terms: ‘cell-free DNA’ and ‘transplantation’. Articles not related to solid-organ transplantation were manually removed (accessed on: 19/10/2021).

Figure 2. Factors influencing cell-free DNA levels.

Table 3. Results of donor-derived cell-free DNA test from a single commercial provider.

Study	Year	Organ	Patients	Cutoff	AUC	PPV %	NPV %	Sensitivity %	Specificity %	Ref.
Bloom et al.	2017	Kidney	102	1.00	0.74	61	84	59.0	85.0	[24]
Huang et al.	2019	Kidney	63	0.74	0.71	77.1	75	79.4	72.4	[68]
Puliyanda et al.	2020	Kidney	67	1.00	0.996	NR	NR	86.0	100.0	[71]
Khush et al.	2019	Heart	740	0.20	0.64	8.9	97.1	44.0	80.0	[69]
Sayah et al.	2020	Lung	69	0.87	0.72	34.1	85.5	73.1	52.9	[72]
Levine et al.	2020	Lung	48	0.51	0.98	NR	NR	81	100	[73]
Khush et al.	2021	Lung	38	0.85	0.67	43.3	83.6	55.6	75.8	[70]

AUC: Area under the curve; NPV: Negative predictive value; NR: Not reported; PPV: Positive predictive value.

Figure 3. Donor-derived cell-free DNA as a transplant biomarker.

(A) Increases in DNA derived from the donor organ were observed following organ damage. Increases are also observed in subclinical disease. (B) Measuring DD-cfDNA levels in transplant recipients longitudinally to screen for: (1) subclinical disease, (2) clinical disease, (3) response to treatment and (4) graft failure.

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