Human Immunology 82 (2021) 850–858

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journal homepage: www.elsevier.com/locate/humimm

Review

Cell-free DNA diagnostics in transplantation utilizing next generation

sequencing
Annette M. Jackson a,⇑, Carly Amato-Menker b, Maria Bettinotti c
a
Duke University, Department of Surgery, DUMC Box 2645, Durham, NC 27710, USA
b
West Virginia University, Microbiology, Immunology, and Cell Biology, Morgantown, WV, USA
c
Johns Hopkins University, Department of Pathology, 2041 E. Monument Street, Baltimore, MD 21205, USA

a r t i c l e i n f o a b s t r a c t

Article history: The use of Next Generation Sequencing (NGS) to interrogate cell-free DNA (cfDNA) as a transplant diag-
Received 14 April 2021 nostic provides a crucial step in improving the accuracy of post-transplant monitoring of allograft health.
Revised 29 June 2021 cfDNA interrogation provides a powerful, yet minimally invasive, biomarker for disease and tissue injury.
Accepted 12 July 2021
cfDNA can be isolated from a variety of body fluids and analyzed using bioinformatics to unlock its ori-
Available online 30 September 2021
gins. Furthermore, cfDNA characteristics can reveal the mechanisms and conditions under which it was
generated and released. In transplantation, donor-derived cfDNA monitoring provides a tool for identify-
Keywords:
ing active allograft injury at the time of transplant, infection, and rejection. Multiple detection and inter-
Transplantation
Rejection
rogation methods for cfDNA detection are now being evaluated for clinical validity and hold the promise
Cell free DNA to provide minimally invasive, quantitative, and reproducible measures of allograft injury across organ
DNA sequencing types.
Biomarker Ó 2021 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights
reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 850
2. Characteristics of circulating cfDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851
3. Collection and processing of Cir-cfDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 852
4. Detection of donor derived Cir-cfDNA as a transplant rejection diagnostic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 853
5. Deciphering the cellular origins of Cir-cfDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 854
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856
Declaration of Competing Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 856

Abbreviations: ABMR, antibody mediated rejection; ACR, acute cellular rejec-

tion; AR, acute rejection; AUC, area under the curve; bp, nucleotide base pairs;
cfDNA, cell-free DNA; dd-Cir-cfDNA, donor derived circulating cell-free DNA; CV,
coefficient of variance; DSA, donor-specific HLA antibody; HHV4-8, human
herpesviruses types 4-8; NPV, Negative predictive values; NGS, Next Generation
DNA Sequencing; PCR, polymerase chain reaction; PPV, positive predictive values;
STR, short tandem repeat; SNP, single nucleotide polymorphism; TCMR, T cell
mediated rejection.
⇑ Corresponding author.
E-mail address: annette.m.jackson@duke.edu (A.M. Jackson).
1. Introduction
Cell-free DNA (cfDNA) are fragments of degraded DNA released
primarily from cells undergoing apoptosis. Due to its rapid turnover,
cfDNA circulating in blood (Cir-cfDNA) provides a window into the
physiology and pathology of cells or tissues in real time. Cir-cfDNA
analysis has been adopted as a valuable clinical laboratory

https://doi.org/10.1016/j.humimm.2021.07.006
0198-8859/Ó 2021 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
A.M. Jackson, C. Amato-Menker and M. Bettinotti
Fig. 1. Cell-free DNA nomenclature and diagnostic utility. Cell-free DNA can be isolated from a variety of human body fluids and utilized for different diagnostic purposes.
This diversity led the 10th International Circulating Nucleic Acids in Plasma and Serum consortium to propose common nomenclature. This figure is being used with author’s
permission [7].
diagnostic and monitoring tool across diverse disciplines such as
oncology, prenatal screening, infectious disease, and transplantation.
The common thread is the need for precision medicine and non-
invasive sampling to allow for accurate and frequent monitoring.
cfDNA can be isolated from a variety of human body fluids such
as blood, urine, saliva, lymph, breastmilk, bile, spinal and amniotic
fluid and serves as a non-invasive biomarker for disease, infection,
and tissue injury [1–5]. Moreover, detectable cfDNA may originate
from self or from non-self sources such as fetal tissue, microbial
and viral DNA, or transplanted stem cells and solid organ allografts.
All individuals harbor baseline levels of cfDNA that derive primar-
ily from ongoing cell turn-over, however the quantity and compo-
sition of cfDNA can vary. Daily activities such as exercise have been
shown to impact circulating plasma proteins levels to include Cir-
cfDNA [6]. Importantly, interrogation of cfDNA characteristics
holds the potential to reveal the source and the mechanisms and
conditions under which cfDNA is generated and released [7]. This
diversity in cfDNA profiles has led members of the 10th Interna-
tional Circulating Nucleic Acids in Plasma and Serum consortium
to recommend a unified cfDNA terminology and nomenclature to
clarify the source of cfDNA in publications and clinical reporting
Fig. 1 [8]. Through bioinformatic analysis of cfDNA, laboratories
can unlock the cell origins and the state of tissue health to advance
non-invasive, personalized medicine.
In the field of solid organ transplantation, detection of donor
derived circulating cfDNA (dd-Cir-cfDNA) is being explored as a
non-invasive measure of allograft injury Fig. 2. There is a growing
851
Human Immunology 82 (2021) 850–858
appreciation for the need for more sensitive methods to diagnose
rejection at its earliest stages prior to clinical allograft dysfunction.
Histopathology remains the gold standard for rejection diagnoses
and provides important phenotypic and cumulative injury per-
spectives. However, currently the detection of early subclinical
rejection requires monitoring through serial surveillance biopsies,
the majority of which reveal no evidence of rejection. The develop-
ment of non-invasive diagnostic tools such dd-Cir-cfDNA assays
may provide a complementary tool for detecting early, subclinical
rejection and also provide information regarding rejection type
(antibody mediated rejection (ABMR) versus T cell mediated rejec-
tion (TCMR)) to guide appropriate interventions.
This review will explore our current understanding of Cir-
cfDNA characteristics and current practices for isolation and inter-
rogation using next generation sequencing (NGS) platforms. We
examined the current literature for clinical studies utilizing Cir-
cfDNA in the setting of transplantation to highlight the current
strengths and limitations of this methodology. Finally, looking to
the future, we discuss how Cir-cfDNA assays may evolve to reveal
greater details surrounding allograft injury and aid in the complex
differential diagnosis of allograft rejection.
2. Characteristics of circulating cfDNA
The concentration of Cir-cfDNA in healthy individuals is low
and a recent study of 84 healthy subjects revealed a range of 1–
10 ng/ml in plasma, with an average of 6 ng/ml reflecting a geno-
A.M. Jackson, C. Amato-Menker and M. Bettinotti Human Immunology 82 (2021) 850–858

Fig. 2. Detection of donor circulating cell-free DNA in transplant diagnostics. Detection of donor derived circulating cfDNA (dd-Cir-cfDNA) is being explored as a non-invasive,
diagnostic measure of allograft injury. Informative single nucleotide polymorphism (SNP) differences between recipient and donor genotypes can be leveraged to allow the
detection and quantification of dd-Cir-cfDNA. In plasma, the majority of Cir-cfDNA molecules derive from the recipient (blue) and a minor fraction derive from donor cells
(black). Detection of increased levels of dd-Cir-cfDNA above baseline indicates allograft injury. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

mic DNA equivalent of 1000 cells [9]. The half-life of Cir-cfDNA in
plasma is short and estimated to be between 10 min and 2.5 h [18].
Removal is facilitated primarily by the liver, but nuclease activity,
degradation by splenic macrophages, and renal excretion also play
a role [7]. Detection of elevated levels of Cir-cfDNA therefore
reflects an active and excessive release of DNA caused by extensive
cell death, inefficient removal of the dead cells, or a combination of
both.
Cell apoptosis is considered the primary source of Cir-cfDNA
within plasma or serum and this is consistent with paired-end
sequencing data showing the dominant DNA species being
nucleosome-size DNA fragments comprising 160–180 base pairs
(bp) [10,11]. Nucleosome packaging of DNA involves 147 bp
wrapped around a histone octamer with an additional linker
DNA of approximately 20–90 bp [12]. This nucleosomal structure
stabilizes DNA in the circulation and provides protection from
enzymatic degradation. Larger Cir-cfDNA fragments have been
reported and are speculated to come from necrotic tissue, while
mitochondrial DNA is also found in the Cir-cfDNA fraction and
the size distribution of this non-histone-bound form is consistently
shorter [10].
The mean concentration of Cir-cfDNA has been reported to be
higher in healthy men when compared to healthy women, which
may reflect differences in body mass [13]. Importantly, patients
with autoimmune disease, cancer, recent transplants, or pregnancy
exhibit elevated Cir-cfDNA compared to healthy individuals
reflecting their altered medical state and/or tissue injury [10,14].
Interestingly, paired-end sequence analyses of Cir-cfDNA mole-
cules deriving from liver allografts have been found to be slightly
smaller (110–140 bp) than predominant Cir-cfDNA molecules
derived from the recipient (165 bp) [15]. Similarly, fetal derived
Cir-cfDNA molecules tend to be smaller (143 bp) than prominent
166 bp maternal fragments [16] and tumor associated Cir-cfDNA
molecules smaller (134–144 bp) than Cir-cfDNA from healthy con-
trols (167 bp) [10,12]. Given that hematopoietic derived blood cells
contribute the majority of the plasma Cir-cfDNA, the smaller Cir-
cfDNA fragments observed from transplants, tumors, and fetal tis-
sue likely reflect tissue-specific differences in nucleosome wrap-
ping which may influence Cir-cfDNA fragment lengths deriving
from different tissues [12,14,15].
3. Collection and processing of Cir-cfDNA
The sensitivity needed for Cir-cfDNA interrogation in many clin-
ical settings requires considerable attention to technical details
surrounding sampling and processing procedures. One of the pos-
sible sources of interference is shedding of genomic DNA from
blood cells after sample collection. Plasma is the preferred Cir-
cfDNA source over serum due to this contamination of genomic
DNA from leukocytes during the clotting process. Raymond et al.
examined the impact of time between blood collection and plasma
processing using replicate samples spiked with a Cir-cfDNA stan-
dard and processed over 7 days. Blood was collected using stan-
dard ethylenediaminetetraacetic acid (EDTA) anticoagulant. This
study showed that longer processing times did not significantly
interfere with the detection of the targeted Cir-cfDNA. A second
study of 106 normal controls using whole blood stored in EDTA
tubes at +4 °C showed no change in Cir-cfDNA concentration after
one day of storage, validating the potential for overnight ship-
ments of samples in this preservative [17]. Nevertheless, in the

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A.M. Jackson, C. Amato-Menker and M. Bettinotti
clinical setting, blood collection tubes that contain stabilizers to
prevent cell lysis remain a preferred choice for blood collection.
The extraction method used to isolate Cir-cfDNA from plasma
can also impact the yield and fragment size bias [18–21]. Compar-
isons have shown that extraction procedures specific for Cir-cfDNA
isolation out-perform standard genomic DNA extraction proce-
dures in providing a better representation of smaller DNA frag-
ments from larger plasma volumes (1–4 ml). Details such as the
number and speed of centrifugation steps to isolate Cir-cfDNA
from plasma has been found to be important. An initial low g-
force centrifugation step is performed to separate plasma from
cells and a second, higher g-force centrifugation removes all
remaining cellular debris. The speed of the second centrifugation
step may influence recovery of different nucleic acid types. Higher
centrifugation speeds (e.g., 16,000
g) aid in removal of intact
genomic DNA from ruptured blood cells, but could also remove
large extracellular vesicles that may contain RNA and cfDNA. Page
et al. examined different kits and different speeds for the second
centrifugation step (1,000
g, 2,000
g, or 10,000
g for 10 min at
4 °C) and found yields of cfDNA to be equivalent, as assessed by
locus-specific TaqMan quantitation of GAPDH, while miRNA yield
was lower after centrifugation at 10,000
g [19]. For clinical diag-
nostics, the extraction procedure, collection tube, and minimal
plasma starting volumes should be optimized for the specific
detection method, analyte, and within specific study populations.
DNA quantification and input for library construction is also an
important step to ensure the sensitivity in detecting tumor, fetal-
derived, or allograft derived Cir-cfDNA. Alborelli et al. performed
Cir-cfDNA analysis on 114 healthy normal controls to determine
feasibility of cancer screening in populations expected to have
low Cir-cfDNA levels [22]. cfDNA was extracted from between
0.4 and 2.0 ml of plasma using a magnetic beads-based cfDNA iso-
lation kit yielding 1.7 to 30.8 ng/ml of cfDNA. Quantification was
performed using a Qubit Fluorometer followed by
electrophoresis-based size selection (Agilent High Sensitivity
D1000 ScreenTape System). Adequate Cir-cfDNA quantity and
quality to proceed to NGS library preparation was achieved for
only 55 out of 114 normal controls. NGS libraries were prepared
using a broad range of cfDNA input (2.5–105.5 ng), with acceptable
results in the lower concentrations, which were far below the min-
imal manufacturers’ recommended amount of 10 ng.
Double-strand library preparation is standard for Cir-cfDNA
analysis because of efficiency, cost, and the optimization of meth-
ods to avoid adapter ligation bias. However, for some applications
such as the paleontological field working with ancient DNA, the
value of single-strand library preparation to recover damaged
and short double-strand DNA fragments, normally lost in double-
strand library preparation, has proven important [23]. Bennet
et al. optimized a single-strand library method using a 50 -
phosphorylated and biotinylated adapter oligonucleotide and a
single-strand DNA ligase to capture single-strand DNA molecules
onto beads without prior end repair. A primer generated comple-
mentary strands, followed by a second adaptor addition via
blunt-end ligation. The molecules are finally heated to release
the finished single strand, which is used in the adaptor sequence
amplification reaction. Cir-cfDNA molecules that are damaged,
for example with single-strand breaks on one or both strands,
are retained within this single-strand library. In a study utilizing
Cir-cfDNA isolated from cancer patients, Sachez et al. compared
double-strand and single-strand library preparations and found
nearly half of the total Cir-cfDNA fragments were<130 nucleotides,
ranging from 30 to 130, and were not detectable using the double-
strand library preparations [12]. Given that Cir-cfDNA fragmenta-
tion may be variable depending on the disease state, optimal
recovery of smaller Cir-cfDNA molecules may be important to
ensure sensitivity and specificity in clinical diagnostics.
853
Human Immunology 82 (2021) 850–858
4. Detection of donor derived Cir-cfDNA as a transplant
rejection diagnostic
The advancement of molecular diagnostic techniques and bioin-
formatics has led to an evolution of methods to detect and charac-
terize Cir-cfDNA within the transplant setting [3,24–27]. Early
studies utilized multiplex, quantitative polymerase chain reaction
(PCR), followed by the development of digital drop microfluidics
PCR and NGS methods. Development of sensitive and efficient
methods for detecting donor derived Cir-cfDNA (dd-Cir-cfDNA)
released from transplanted allografts promises to provide a mini-
mally invasive, quantitative, and reproducible measure of allograft
injury across organ types.
Transplanted allografts are injured at multiple points during the
transplant process and it is the cumulative injury that likely
impacts long-term survival potential [28–33]. Peri-operatively
allografts sustain injury during procurement, cold storage, and at
time of reperfusion. Acute rejection is also common, particularly
within the first year post-transplant. Infection is likely an underap-
preciated form of allograft injury except in cases of severe infection
coinciding with allograft dysfunction. A multidisciplinary inves-
tigative group at Stanford University established a ‘‘shotgun” or
random DNA NGS sequencing approach to monitor dd-Cir-cfDNA
in heart and lung transplant recipients by quantifying tens-of-
thousands of reads containing informative single nucleotide poly-
morphism (SNPs) differentiating donor from recipient [34]. The
estimated informative SNP variations between individuals is 1.6
million, using sites where the recipient is homozygous and the
donor possesses a different nucleotide on one or both or alleles
[35]. Each sequencing read containing an informative SNP is char-
acterized as recipient, donor, or PCR artifact and from these data
the percentage of dd-Cir-cfDNA to recipient Cir-cfDNA is calcu-
lated. Therefore, for these studies both the recipient and donor
genotypes are needed to distinguish donor-specific sequencing
reads. Analyses of longitudinal post-transplant plasma samples
obtained from heart and lung recipients showed high levels of
dd-Cir-cfDNA on the first day post-transplant, followed by an
exponential decay to a low, steady state baseline level [36,37].
Organ-specific differences were observed in the amount of dd-
Cir-cfDNA detected immediately post-transplant and in the decay
kinetics. Consistent with organ mass, dd-Cir-cfDNA was higher in
bilateral compared to single-lung transplants and higher in lung
transplants compared to heart transplants. The heart transplant
data showed single decay kinetics (single exponential), while the
lung transplant data showed a two-step decay model during the
first weeks post-transplant.
Studies have also demonstrated elevations in dd-Cir-cfDNA
levels above baseline occurring at time of infection [36–39]. Ran-
dom shotgun sequencing allows data interrogation for pathogen-
derived sequences and extends the scope of Cir-cfDNA monitoring
to include infectious disease detection. For lung recipients
elevated dd-Cir-cfDNA correlated with clinically diagnosed cyto-
megalovirus infection in serum or lung lavage fluid with an area
under the curve (AUC) = 0.91 [37]. High frequency clinical and
subclinical infections were also detected involving polyomavirus,
human herpesviruses (HHV4-8) and adenovirus as well as bacte-
rial and fungal infections, some of which are not routinely tested
for post-transplant.
Detection of acute rejection in the early subclinical stages, prior
to injury sufficient to yield dysfunction, and early identification of
chronic rejection lesions would provide opportunities for interven-
tion in the hopes of improving long-term allograft survival. The
current ‘‘gold standard” diagnostic for acute and chronic rejection
remains the histological grading of biopsy tissue. However,
histopathology has inherent limitations relating to sensitivity,
inter-observatory variability, and dependence on qualitative not
A.M. Jackson, C. Amato-Menker and M. Bettinotti
quantitative measures. Therefore, determining clinical validity of
more sensitive and potentially more accurate rejection diagnostics
will require rigorous validation studies using long-term allograft
function and survival as clinical endpoints. Agbor-Enoh et al. inde-
pendently validated and applied the Stanford Shotgun NGS method
using a 1% threshold for dd-Cir-cfDNA and longitudinal samples
from lung and heart recipients [32,38,40,41]. These studies added
to previous reports showing the utility of dd-Cir-cfDNA to detect
injury at time-points preceding clinical histopathological diag-
noses for rejection and before clinical dysfunction [36,37]. The
Stanford collaborative group have further developed a bioinfor-
matics algorithm to identify minority donor sequencing reads
and thereby eliminate the need for full recipient and donor
genotype information [42]. Nevertheless, removal of the actual
donor and recipient genotype information from the bioinformat-
ics increases the need to guard against false positive results
stemming from blood products or cases of multi-organ or
re-transplantation.
The current commercial assays including those in clinical trials
use multiplex PCR coupled with NGS to specifically target hun-
dreds to thousands of informative SNPs to detect dd-Cir-cfDNA.
In these assays, the predominant, informative SNPs are assigned
to the recipient and the minority fraction are arbitrarily assigned
as dd-Cir-cfDNA using algorithms that do not require recipient or
donor genotypes. Grskovic et al. reported on the precision of dd-
Cir-cfDNA detection using a NGS targeted sequencing of 266 infor-
mative SNPs [43]. Assay validation utilized 1,117 samples from the
National Institute for Standards and Technology Genome in a Bot-
tle human reference genome and determined a limit of detection of
0.16% and limit of quantification of 0.20% dd-Cir-cfDNA. The linear
quantifiable range was 0.2%  16% and run-to-run coefficient of
variance (CV) was 6.8%. Bromberg et al. reported further using this
same assay to examine dd-Cir-cfDNA biological variation within
380 longitudinal blood samples from 93 stable transplant recipi-
ents [44]. The median dd-Cir-cfDNA was 0.21% (interquartile range
0.12%–0.39%) and 96% of the samples exhibited values below 1.0%.
Of note, these studies were performed using a centralized labora-
tory and further interlaboratory studies will be needed if siimilar
assays are released to transplant laboratories in a kit form.
Retrospective and prospective clinical studies have been per-
formed in kidney, heart, lung, and liver transplant cohorts and
are summarized in Table 1 [36,37,39,41,45–57]. Rejection preva-
lence and rejection type varied between studies and different %
dd-Cir-cfDNA positive thresholds were used depending on the
study endpoint. In spite of these variations, some common patterns
have emerged. In general, higher dd-Cir-cfDNA thresholds were
sufficient to detect active ABMR whereas lower thresholds were
needed to correlate with clinical and borderline TCMR. Negative
predictive values (NPV) were high and ranged from 83 to 100%
across studies and organ types; however, the positive predictive
values (PPV) were lower in all cases and varied widely between
studies Table 1.
NPV and PPV describe the clinical validity of dd-Cir-cfDNA diag-
nostic tests, using a set % threshold for positivity and expected
rejection prevalence Fig. 3. NPV represents the specificity of a
screening test to detect all true negatives while PPV estimates
the sensitivity to correctly identify all recipients who have allograft
injury (true positives) [58]. Interpreting these values in the setting
of clinical transplantation requires full recognition of the current
limitations to our gold standard (histopathology) for rejection
diagnoses and our current limitations to detect all sources of allo-
graft injury (e.g. subclinical rejection, infection and disease recur-
rence). The lower PPVs in all of these clinical studies may reflect
methodology problems with the assays or misconceptions sur-
rounding the full range of insults that an allograft incurs, making
it difficult to accurately assess the clinical validity of dd-Cir-
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Human Immunology 82 (2021) 850–858
cfDNA across diverse study cohorts. The prevalence of rejection
in each cohort can impact the diagnostic acumen of the assay;
increased rejection prevalence in higher risk cohorts will increase
the PPV but decrease the NPV. Similarly, lower rejection prevalence
decreases the PPV while increasing the NPV [58]. Given the low
PPV and current limitations in our understanding of how to inter-
pret and whom and when to monitor for dd-Cir-cfDNA, this assay
should not be considered a stand-alone diagnostic. Rather the high
NPV signifies utility as an integrated screening test to be inter-
preted with other clinical parameters, immunologic risk categories,
and companion diagnostics from infectious disease and histocom-
patibility laboratories.
Newer studies are evaluating dd-Cir-cfDNA measurements calcu-
lated as absolute concentrations (cp/mL) versus the conventional per-
cent fraction (%) of recipient Cir-cfDNA. Zhao et al. measured dd-Cir-
cfDNA in 49 pediatric liver recipients and observed similar correla-
tions with biopsy proven rejection (n = 11) whether using absolute
concentration (AUC = 0.841) or percent fraction (AUC = 0.878) [39].
In contrast, Oellerich et al. performed a longitudinal analysis of 189
kidney recipients and showed that the absolute concentration of
dd-Cir-cfDNA (AUC = 0.83), when detected using digital droplet
PCR, outperformed the percent fraction (AUC = 0.73) in discriminating
rejection (n = 15) from stable allograft function [59]. One potential
reason for this latter finding and a potential advantage for calculating
absolute concentrations is the finding that exercise can increase Cir-
cfDNA derived from hematopoietic cells in the blood, resulting in
higher recipient cfDNA levels and a potential underestimation of
dd-Cir-cfDNA levels when using a percent fraction calculation [20].
Two studies, in kidney and lung, show the power of combining
dd-Cir-cfDNA and HLA antibody detection methods. Jordan et al.
investigated the utility of dd-Cir-cfDNA to aid in ABMR diagnoses
when combined with donor-specific HLA antibody (DSA) testing
[48]. Eighty-seven kidney recipients were examined using 90 blood
samples with paired DSA and biopsies performed due to clinical
suspicion. The PPV of dd-Cir-cfDNA (1% positive threshold) to
detect active ABMR in 16 DSA positive patients was 81% and the
NPV was 83%. In comparison, the ABMR PPV for DSA + alone was
48%. This study highlights the use of multiple biomarkers to
increase diagnostic accuracy. dd-Cir-cfDNA is a sensitive short-
lived biomarker of allograft injury, but is not specific for rejection.
DSA holds a much longer half-life (~30 days) in the blood and sig-
nifies an active alloimmune response, but at low levels DSA may
not correlate with significant tissue injury. Agbor-Enoh et al. also
examined the relationship between DSA and dd-Cir-cfDNA in 34
lung recipients with active ABMR. All patients had detectable
DSA at time of ABMR diagnosis exhibiting a wide MFI range
(1896–24,278). Using mixed linear modeling, a significant correla-
tion was seen between DSA MFI and dd-Cir-cfDNA levels [32].
Therefore, utilizing dd-Cir-cfDNA in combination with DSA detec-
tion may enhance the detection of clinical and subclinical ABMR.
Furthermore, enhancements in dd-Cir-cfDNA specificity, as
described in the next section, may increase diagnostic potential
for subclinical injury that occurs in the absence of clinical dysfunc-
tion or histopathological evidence of rejection.
5. Deciphering the cellular origins of Cir-cfDNA
Future improvements for Cir-cfDNA diagnostics in transplanta-
tion will include the ability to trace this biomarker back to the
exact cell types that have been injured and released DNA. This
advancement would broaden the utility of Cir-cfDNA as a mulit-
purpose diagnostic in transplantation, beyond detecting rejection
alone. This capability would provide considerable clarity surround-
ing the type and mechanism of allograft injury such as antibody
mediated injury of peritubular capillaries versus podocyte injury
A.M. Jackson, C. Amato-Menker and M. Bettinotti Human Immunology 82 (2021) 850–858

Table 1

Organ type Cohort Rejection prevalence Endpoint Threshold & AUC PPV NPV
Kidney
Bloom, RD et al., J Am Soc N = 102 Biopsy-paired samples = 107 No rejection Threshold 1% AR = 61% =84%
Nephrol, 2017 AR = 27 cases Vs AR AR AUC = 0.74 ABMR = 44% =96%
ABMR = 16; TCMR = 11 Vs ABMR ABMR AUC = 0.87
TCMR = ND
Jordan SC, et al., Transplantation N = 87 Biopsy-paired sample & DSA = 90 DSA+ ABMR Threshold 1% ABMR = 81% =83%
Direct 2018 DSA+ ABMR = 16 cases
Sigdel TK, et al., J Clin Med, 2019 N = 193 Biopsy-paired samples = 217 No rejection Threshold 1%
AR = 38 cases Vs AR AR AUC = 0.87 AR = 51.9% = 95.1%
Borderline AR = 72 cases Borderline AR = No
correlation
Huang E, et al., Am J Transplant, N = 63 Cross sectional No rejection Threshold = .74% ABMR = 68.6% = 100%
2019 analysis = suspicious for rejection Vs TCMR TCMR AUC = 0.42
AR+ = 34 cases Vs ABMR ABMR AUC = 0.82
DSA+ = 27 cases
Gielis EM, et al., Nephrol Dial N = 107 Biopsy-paired samples = 65 No rejection Threshold = 0.88% AR = 8.7% = 97.8%
Transplant, 2020 AR = 13 cases Vs AR AR = AUC 0.64
Stites E, et al., Am J Transplant. N = 79 Cross sectional analysis= eGFR decline Threshold 0.5% NA NA
2020 TCMR 1A & Borderline = 79 cases low cf-DNA <0.5% Multivariate analysis
Vs 6 month -8.5% eGFR decline
High cfDNA >0.5% p = .0040
Puliyanda DP, et al., Ped N = 67 ped Cross sectional analysis = 48 No rejection Threshold 1% NA NA
Transplantation, 2020 cases Vs AR AR AUC = 0.996
suspicious for rejection
OR biopsies w/ dd-cfDNA >1.0%
Zhang H, et al. FrontImmunol N = 37 18=ABMR+, DSA+ No rejection Threshold 1% ABMR = 76.2% = 87.5%
2020 Vs ABMR ABMR AUC = 0.90
Shen J et al. Clin Tranplants, 2020 N = 28 Cross sectional analysis Treatment assoc positive correlation @ NA NA
ABMR = 5 Ddd-cfDNA% respective timepoints
TCMR (1A or1B) = 12 Vs q = 2.570, P = .022
TCMR (2A or 2B) = 11 DeGFR (1,3,6 mo) q = 3.210, P = .027
q = 2.860, P = .019
Heart
De Vlaminck I, et al., Sci Transl N = 21 ped 356 samples paired with No rejection Threshold = .25% NA NA
Med 2014 N = 44 endomyocardial biopsies Vs ACR < 2R/3A Mild AR AUC = 0.63
adult AR = 24 cases Vs ABMR 1 or Mod/Severe AR AUC = 0.83
ACR 2R/3A
Khush KK, et al., Am J Transplant Multi 841 samples paired with No rejection Threshold = .20% ABMR 1 or
2019 Cohorts endomyocardial biopsies Vs ABMR 1 or ACR AUC = 0.64 ACR 2R = 97.1%.
N = 443 >55 days posttransplant 2R AUC = NA = 8.9% = 97.9%
N = 33 ACR = 17 & ABMR = 18 No rejection ABMR 1
High risk 110 plasma samples Vs ABMR 1 = 20.2%
>14 days post-transplant
ABMR = 11
North PE, et al., PLoS One 2020 N = 76 158 samples paired with No rejection Threshold = 0.32% ACR = 43.90% =94.87%
Ped & endomyocardial biopsies Vs ACR 2R AUC = 0.842
Adult 8 days post-transplant
Richmond ME, et al., J Heart Lung N = 241 624 samples paired with No rejection Threshold = 0.16% ACR 1R or
Transplant 2020 Ped & endomyocardial biopsies Vs ACR 1R or AUC = 0.78 ABMR 1 = 85.7%
Adult >7 days post-transplant ABMR 1 = 65.2%
ACR 1 = 15 cases
AMR 1 = 20 cases

Agbor-Enoh S, et al., Circulation N = 171 1,392 endomyocardial biopsies No rejection Threshold = .25% ABMR 1
2021 1,834 plasma samples Vs ABMR 1 or AUC = 0.92 ACR = 99%
ACR 2 = 38 cases ACR 2R 2R = 19.6%
AMR 1 = 32 cases
Mixed AMR/ACR = 3 cases
Lung
De Vlaminck I, et al., Proc Natl N = 51 Biopsy-paired samples = 113 No rejection Threshold 1% NA NA
Acad Sci 2015 >60 days post-transplant Vs severe ACR A3 ACR A3 AUC = 0.90
Vs mild-severe ACR ACR A1 AUC = 0.70
A1
Sayah D, et al., Transplantation N = 69 ACR = 13 No rejection Threshold = 0.87% ACR = 34.1% = 85.5%
Direct 2020 Vs ACR A1 ACR AUC = 0.717
Levine DJ, et al., Biomarker N = 48 CLAD, N = 10 No rejection Threshold = 0.51% NA NA
Insights, 2020 ABMR, N = 9 Vs AR AUC = 0.98
ACR, N = 2
Liver
Zhao D, et al., Scientific Reports N = 49 Biopsy-paired samples = 49 No rejection Threshold = 28.7% AR = 80% = 92.3%
2021 Ped AR=11 Vs AR AUC = 0.878 AR = 56.2% = 93.9%
Threshold = 2076 cp/mL
AUC = 0.841

855
A.M. Jackson, C. Amato-Menker and M. Bettinotti
Fig. 3. Determining analytical and clinical validity of cell-free-DNA diagnostics. Sensitivity measures the proportion of positive tests that correctly identify rejection (True
Positive) while Specificity measures the proportion of negative tests that correctly identify no rejection (True Negative). Negative and Positive Predictive Values (NPV and
PPV) describe the performance of a diagnostic test using a set threshold for positivity and expected rejection prevalence. NPV represents the specificity of a screening test to
detect all true negatives while PPV estimates the sensitivity to correctly identify all recipients who have allograft rejection (true positives).
correlating with recurrent glomerular disease. Cir-cfDNA monitor-
ing may also allow early detection of malignancies in transplant
patients. This could advance the diagnosis of graft-versus-host dis-
ease following hematopoietic stem cell transplantation or multi-
visceral transplants by detecting elevated recipient tissue injury
in the gut and skin. Furthermore, Cir-cfDNA diagnostics could be
extended to the pre-transplant setting for monitoring the progres-
sion of end-stage organ failure or recovery following living organ
donation.
Current clinical diagnostics distinguish Cir-cfDNA origins (self
versus non-self) based on the genetic differences (SNPs). This is
true for prenatal screening (fetal versus maternal origins), mutated
tumors versus heathy tissues, and transplant rejection (donor ver-
sus recipient). However, Cir-cfDNA that is derived from different
cell types often exhibit unique DNA methylation patterns, histone
modifications, and nucleosome positioning reflecting cell-specific
gene expression patterns [60–62]. The lack of genetic differences
(SNPs) within self-DNA can be overcome by interrogating the
cells-specific epigenetic marks reflecting silent genes (DNA methy-
lation) or active genes (histone modifications and nucleosome
positioning). Cells of the same type will share nucleosome posi-
tioning within specific genes; these nucleosome-protected DNA
sequences will undergo less protease digestion in the blood and
will be enriched in the Cir-cfDNA fraction if injury is localized to
one tissue region or cell type. Deep sequencing of Cir-cfDNA can
generate maps of in vivo nucleosome occupancy reflecting the
cell-specific nuclear architecture and gene expression patterns.
These nucleosome footprints can then be used to infer the cell
types or tissue types contributing to Cir-cfDNA in pathological
states such as transplant allograft rejection. This type of specificity
and increased sensitivity could aid in the diagnosis of borderline
acute rejection, ABMR versus TCMR versus mixed rejection, and
potentially differentiate rejection from other forms of injury such
as recurrent disease.
856
Human Immunology 82 (2021) 850–858
6. Conclusion
The addition of dd-Cir-cfDNA detection by NGS as a noninvasive
transplant diagnostic provides a significant opportunity to improve
the detection of rejection, infection, and recurrent disease. Under-
standing the characteristics of Cir-cfDNA impacted by isolation and
processing is crucial to achieve accurate and sensitive results. Cur-
rent commercial assays that quantitate major (recipient) and
minor (donor) Cir-cfDNA fractions are available, yet further studies
are needed to clinically validate specific endpoint thresholds and
determine optimal use with companion diagnostics provided by
infectious disease and histocompatibility laboratories. Future
advances in deciphering the exact origins of Cir-cfDNA will further
improve its utility and accuracy as a diagnostic tool. Importantly,
dd-Cir-cfDNA assays hold the potential for non-invasive monitor-
ing allograft health, providing highly sensitive injury surveillance
in real time, and facilitating early detection with the possibility
of intervention prior to clinical dysfunction. Integrated with clini-
cal parameters, histocompatibility and infectious disease diagnos-
tics, and allograft function tests, the measurement of dd-Cir-cfDNA
increases our arsenal of tools to improve transplant outcomes.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
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