Comparison of Methods for Donor-Derived Cell-Free DNA Quantification in Plasma and Urine from Solid Organ Transplant Recipients

doi:10.21203/rs.3.rs-1536352/v1

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Comparison of Methods for Donor-Derived Cell-Free DNA Quantification in Plasma and Urine from Solid Organ Transplant Recipients

https://doi.org/10.21203/rs.3.rs-1536352/v1

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Background

In allograft monitoring of solid organ transplant recipients, liquid biopsy has emerged as a novel approach using quantification of donor-derived cell-free DNA (dd-cfDNA) in plasma. Despite early clinical implementation and analytical validation of techniques, direct comparisons of dd-cfDNA quantification methods are lacking. Furthermore, data on dd-cfDNA in urine is scarce and high-throughput sequencing-based methods so far have not leveraged unique molecular identifiers (UMIs) for absolute dd-cfDNA quantification.

Methods

Different dd-cfDNA quantification approaches were compared in urine and plasma of kidney and liver recipients: A) Droplet digital PCR (ddPCR) using allele-specific detection of seven common HLA-DRB1 alleles and the Y chromosome; B) high-throughput sequencing (HTS) using a custom QIAseq DNA panel targeting 121 common polymorphisms; and C) a commercial dd-cfDNA quantification method (AlloSeq® cfDNA, CareDx). Dd-cfDNA was quantified as %dd-cfDNA, and for ddPCR and HTS using UMIs additionally as donor copies. In addition, relative and absolute dd-cfDNA levels in urine and plasma were compared in clinically stable recipients.

Results

The HTS method presented here showed a strong correlation of the %dd-cfDNA with ddPCR (R² = 0.98) and AlloSeq® cfDNA (R² = 0.99) displaying only minimal to no proportional bias. Absolute dd-cfDNA copies also correlated strongly (τ = 0.78) between HTS with UMI and ddPCR albeit with substantial proportional bias (slope: 0.25; 95%-CI: 0.19–0.26). Among 30 stable kidney transplant recipients, the median %dd-cfDNA in urine was 39.5% (interquartile range, IQR: 21.8–58.5%) with 36.6 copies/µmol urinary creatinine (IQR: 18.4–109) and 0.19% (IQR: 0.01–0.43%) with 5.0 copies/ml (IQR: 1.8–12.9) in plasma without any correlation between body fluids. The median %dd-cfDNA in plasma from eight stable liver recipients was 2.2% (IQR: 0.72–4.1%) with 120 copies/ml (IQR: 85.0-138) while the median dd-cfDNA copies/ml was below 0.1 in urine.

Conclusions

This first head-to-head comparison of methods for absolute and relative quantification of dd-cfDNA in urine and plasma supports a method-independent %dd-cfDNA cutoff and indicates the suitability of the presented HTS method for absolute dd-cfDNA quantification using UMIs. To evaluate the utility of dd-cfDNA in urine for allograft surveillance, absolute levels instead of relative amounts will most likely be required given the extensive variability of %dd-cfDNA in stable kidney recipients.

dd-cfDNA

HTS

biomarker

ddPCR

kidney

liver

urine

plasma

Solid organ transplantation is widely accepted as the best treatment for various end-stage organ diseases, improving quality of life and reducing mortality. Even though the rate of graft survival has steadily increased over the years in both the short and long term, graft-related pathologies such as antibody- or cell-mediated rejection, infections and drug-related toxicity remain major hurdles to overcome. Improvements have been made regarding diagnostic tools leading to an increased graft survival, however biopsies remain the gold standard for diagnosis of allograft dysfunction. The significant costs, potential complications and inter-observer interpretation variability in allograft biopsies highlight the need for additional biomarkers to detect or exclude acute rejection, ideally at a subclinical stage, and other causes of allograft dysfunctions in a cost-efficient and less invasive manner.

Liquid biopsy has emerged as a novel minimally invasive approach using quantification of cell-free DNA (cfDNA) originating from the allograft, so-called donor-derived cfDNA (dd-cfDNA), in plasma. Large, prospective, multicenter studies [1–5] show the validity of dd-cfDNA to detect acute allograft injury. While most investigations focused on the relative amount of dd-cfDNA among the total cfDNA (%ddcfDNA), recent data [6–9] including large clinical studies suggest a potentially improved diagnostic performance with absolute quantification, i.e. dd-cfDNA copies/ml. Factors influencing the amount of circulating recipient-derived cfDNA such as exercise [10] in the short term, and changes in dynamic immunosuppressive regimens [9] as well as other diseases [11] in the long term, highlight the shortcomings of dd-cfDNA quantification based solely on relative amounts. Furthermore, the majority of research so far has focused on plasma as the source of dd-cfDNA and only few studies have investigated dd-cfDNA in urine. [12] Limited results show potential for urinary dd-cfDNA with improved practicality as a biomarker especially for, but not limited to renal allograft injury. [12]

Different methods have been used for the detection and quantification of total and dd-cfDNA, namely quantitative PCR, droplet digital PCR (ddPCR) and high-throughput sequencing (HTS), which all differ in terms of cost, turnaround time and infrastructure requirements. Depending on the techniques either specific HLA mismatches, sex differences or common single-nucleotide or copy number polymorphisms were used as molecular targets to differentiate donor- and recipient-derived cfDNA. While the first two approaches utilize readily available donor genotype information, and thus, do not require separate donor genotyping or derivation of donor genotypes, the use of common polymorphisms can be applied more broadly independent of the presence of mismatches at individual loci. In recent years, several clinically [4, 13] and analytically [14, 15] validated HTS-based methods have entered the market, which are increasingly used as a non-invasive graft surveillance tool and are reimbursed by Medicare (USA) since October 2017. With clinical adoption, organ-specific %dd-cfDNA cutoffs have been proposed. However, these cutoffs are based on data originating from measurements with different methods. Despite the rise of dd-cfDNA as a non-invasive allograft monitoring approach, studies directly comparing methods, molecular targets and in different body fluids are lacking.

Here we present a direct head-to-head comparison of different analytical methods for the absolute and relative quantification of dd-cfDNA in urine and plasma from kidney and liver transplant recipients. Given the potential benefit of simultaneous dd-cfDNA quantification both as relative and absolute amounts, we developed a novel HTS-based method that incorporates unique molecular identifiers (UMIs) and evaluated its potential for quantification of dd-cfDNA copies. Furthermore, we also investigated relative and absolute levels in urine and plasma in stable solid organ recipients and the correlation between both body fluids.

2.1. Patient Recruitment and Sample Collection

Patients were recruited at the Inselspital (Bern University Hospital, Switzerland) between August 2019 and August 2020. The study was approved by the ethics committee of the Canton of Bern, Switzerland (2019 − 00730). From liver and kidney transplant recipients who provided informed consent, whole blood and urine samples were collected once at a routine follow-up appointment or at up to three time points within the first week after transplantation. For data analysis, kidney transplant recipients were categorized as stable based on normal renal biopsy results and, in absence of biopsy results, on low average serum creatinine levels. Liver transplant recipients were considered stable if total bilirubin, aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) levels were within the normal range. In addition, anonymized plasma samples from 20 healthy volunteers without a history of organ transplantation, no recent blood transfusion or pregnancy were utilized.

Venous blood samples were collected in two 7.5 ml K3 EDTA blood collection tubes (Sarstedt, Nümbrecht, Germany) and urine was collected in a 50 ml LoBind® tube (Eppendorf SE, Hamburg, Germany). The Cell-Free DNA Urine Preserve (Streck Inc., La Vista, NE, USA) was added to urine samples to prevent cfDNA degradation and release of cellular genomic DNA (gDNA). For all downstream sample processing or storage steps, LoBind® tubes were used.

EDTA whole blood samples were centrifuged within 2 hours of collection at 2’000g for 15 minutes at room temperature (RT). The plasma was re-centrifuged at 3’800g for 10 minutes at RT. The full plasma supernatant and up to 1ml of buffy coat were stored at -20°C until DNA extraction. The urine was centrifuged at 3’000g for 15 minutes and the supernatant was initially stored at -20°C or -80°C.

2.2. cfDNA and gDNA Extraction

The plasma was thawed at 4°C and cfDNA was extracted with the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany), according to the protocol provided by the manufacturer. The 5 ml extraction scheme was applied using an elution volume of 150 µl and a second elution step by reapplying the eluate to the elution column. The eluate was subsequently concentrated using Amicon® Ultra-0.5 30K Centrifugal Filter Devices (Merck, Darmstadt, Germany) according to the instructions provided by the manufacturer with a centrifugation time of 3.5 min.

The urine samples were thawed at RT and the cfDNA was isolated using the Quick-DNA Urine Kit (Zymo Research, Irvine, CA, USA). The extraction was performed according to the “Cell-free DNA only” protocol provided by the manufacturer with a prolonged incubation with proteinase K at 55°C for 60 minutes instead of 30 minutes to ensure optimized protein digestion and increased cfDNA yield. Elution was performed with 40 µl DNA Elution Buffer pre-heated to 65°C and with a re-elution step.

The eluate volume was determined with a 10–100 µl pipette (Eppendorf SE) and the DNA concentration was measured using the Qubit® 1X dsDNA HS Assay Kit and Qubit® 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The isolated plasma-derived and urinary cfDNA was subsequently stored at -80°C until further analysis.

2.3. Spike-in cfDNA Dilution Series

Two cfDNA spike-in dilution series were prepared, each using cfDNA extracted from plasma of two healthy volunteers. Approximately equal fragment size distributions were ensured using the High Sensitivity DNA Kit with the 2100 Bioanalyzer system (Agilent, Santa Clara, CA, USA). The mixture with the highest percentage of spike-in cfDNA used for the linearity assessment of the HTS method was also measured with ddPCR. The fractional abundance obtained by ddPCR was then used for calculation of the theoretical percentage spike-in of all samples in the series based on the dilution factor used.

2.4. Dd-cfDNA Quantification by ddPCR

2.4.1. Assays and Validation

For the detection and quantification of dd-cfDNA by ddPCR, assays with sequence-specific TaqMan hydrolysis probes were used targeting alleles of the HLA-DRB1 gene [16]. Of the eight previously described assays, the following seven were ordered from Bio-Rad Laboratories Inc., Hercules, CA, USA, with HEX or FAM fluorophores, based on the high frequencies of the targeted alleles in the Swiss population: HLA-DRB1*01 (dHsaEXD29156242), HLA-DRB1*03 (dHsaEXD93426015), HLA-DRB1*04 (dHsaEXD10134188), HLA-DRB1*07 (dHsaEXD74403961), HLA-DRB1*11 (dHsaEXD22943507, dHsaEXD80505107), HLA-DRB1*13 (dHsaEXD54774880) and HLA-DRB1*15 (dHsaEXD29044653). Of note, the assay for the HLA-DRB1*03 allele was also specific for the gene and allele HLA-DRB3*03, while the HLA-DRB1*15 assay was also specific for the HLA-DRB1*16 allele. In addition, an assay targeting the AGO1 gene was also used with both HEX (dHsaCP2500349) and FAM (dHsaCP1000484) fluorophores for the quantification of haploid genomes present in a sample.

To quantify cfDNA from male donor organs in female recipients, two assays targeting the Y-chromosomal SRY gene were used. For one, primers (forward: 5’ – TGTCCTACAGCTTTGTCCAG – 3’; reverse: 5’ – CCACTTACCGCCCATCAAC – 3’) and a probe (5’ – FAM-ACCGCAGCAACGGGACCGCT-BHQ-1–3’ were newly designed for amplification under identical conditions as the DRB1 alleles with an amplicon length of 77 bp. Primers and probe were ordered from Microsynth AG, Balgach, Switzerland and combined to a 20x concentrated assay mix with a final concentration in the reaction mixture of 450 nM for both the forward and reverse primers and 250 nM for the probe. Additionally, the ddPCR assay (dHsaCP2500472) targeting the SRY gene from Bio-Rad Laboratories Inc. was used.

To perform limited assay validation in addition to the published data [16], homozygous reference DNA samples from the Consanguineous Reference Panel were ordered from the International Histocompatibility Working Group (Fred Hutchinson Cancer Research Center, Seattle, WA, USA). These DNA samples were first amplified using whole genome amplification of 100 ng DNA per sample with the REPLI-g Mini Kit (Qiagen) following the protocol provided by the manufacturer and subsequently digested with HindIII (Promega Corp., Madison, WI, US) with 5 U of enzyme, for 2 hours at 37°C. The DNA was subsequently quantified and diluted to a concentration of about 10 ng/µl. The ddPCR assays were tested with the corresponding reference target HLA-DRB1 allele and non-target reference samples to ensure the absence of unspecific amplification. Further, the same tests were performed with cfDNA extracted from plasma of HLA typed healthy individuals.

2.4.2. Experimental Setup and Measurement

Each ddPCR reaction simultaneously contained one donor-specific assay and either a recipient-specific assay or an assay targeting both donor and recipient, using probes with different fluorophores. For each cfDNA sample, whenever possible, two different donor/recipient/total cfDNA assay combinations were measured per sample as triplicates, ideally one combination targeting donor- and recipient-specific HLA-DRB1 alleles, and one combination targeting AGO1 and a donor-specific HLA-DRB1 allele or SRY. In samples where no two different assay combinations were available, two triplicates of the same assay combination were measured.

For ddPCR analysis, the QX200 AutoDG Droplet Digital PCR System (Bio-Rad Laboratories Inc.) was used and performed according to the manual “ddPCR® Copy Number Variation Assays, Validated” (#10033173 Ver C) with the previously published PCR protocol [16] with an annealing temperature at 55°C. Up to 25 ng cfDNA were used per reaction. The QX200 droplet reader (Bio-Rad Laboratories Inc.) with the two-color detection system set to FAM/HEX and in the rare event detection (RED) mode was used for data acquisition.

2.4.3. ddPCR Data Analysis

The QuantaSoft software v1.7.4 (Bio-Rad Laboratories Inc.) was used to analyze the ddPCR data. The measured copies were adjusted for the proportion of non-amplifiable copies as described below. In addition, the number of copies for alleles present with two copies per genome was adjusted to a single copy per diploid genome. The mean recipient- and donor-derived cfDNA copies/ml of the triplicates were calculated and used to calculate the mean for both assay combinations. For assay combinations, where the target was present in both the recipient and donor such as AGO1 or matching HLA-DRB1 alleles, the donor or recipient copy numbers were only measured indirectly and were determined by subtraction. Specifically, the copies of the target, which was only present in the recipient or donor, were subtracted from the copies of the target present in both the recipient and donor simultaneously, considering the zygosity of the targets.

2.4.4. Adjusting for Non-Amplifiable Fragments and Concentration of Urine

To correct for the proportion of target cfDNA fragments that were not PCR-amplifiable because they did not contain both PCR primers of the used assay, the formula (Eq. 1) proposed by Oellerich et al. [7] was used. Instead of a sample-specific mean fragment length, a body fluid-specific fragment length was used (186 bp for plasma, 208bp for urine), which was determined as described in the supplementary methods (Additional file 1).

Equation 1: Adjusting for the proportion of non-amplifiable fragments. Variables include the mean fragment length and the assay-specific amplicon length.

For measurements in urine, dd-cfDNA copies/ml were also normalized by the urinary creatinine (UCr) level at the time (± 1.5 h) of urine collection because the concentration of urine can vary depending on the patient’s fluid intake.

2.5. Dd-cfDNA Quantification by QIAseq HTS

2.5.1. Assay Design and Validation

For the detection and quantification of dd-cfDNA with HTS, a custom QIAseq Targeted DNA Panel (Qiagen) was used, comprised of 121 common, disease-unrelated SNPs chosen based on their high population MAF (Additional file 3). A total of 115 SNPs included in the panel were distributed over all 22 autosomes with an additional three SNPs for each the X and the Y chromosome. To maximize coverage for all targets considering the fragmentation of cfDNA, two specific primers (one on each strand) were designed for each target with the support of the QIAGEN bioinformatics team. The QIAseq protocol incorporates unique molecular identifiers (UMI) during library preparation, enabling the elimination of PCR amplification bias relevant in absolute quantification and low abundance variant detection with HTS.

2.5.2. Library Preparation and Sequencing

Libraries were prepared using 10–40 ng cfDNA and sequenced according to the manual of the QIAseq Targeted DNA kit with 18 universal PCR cycles and using the QIAseq 96-Unique Dual Index Set A (Qiagen) to prevent index cross-talk. The library concentration was determined with the Qubit™ dsDNA 1x HS Assay Kit (Thermo Fisher Scientific) and the libraries were stored at -20°C until pooling. The sequencing pool was combined on the day of sequencing at a concentration of 4 nM using a mean library fragment size of 315 bp and subsequently stored at 4°C until denaturation. The pool was paired-end sequenced on an Illumina NextSeq 500 (Illumina, San Diego, CA, USA) with a NextSeq 500/550 Mid Output Kit v2.5 (300 Cycles). To generate 3–5 reads/UMI, about 6 million reads were sequenced per sample.

2.5.3. Bioinformatic Analysis

Demultiplexing was executed using the bcl2fastq Conversion Software v1.8.4. The resulting FASTQ files were imported into the CLC Genomic Workbench (v20) (Qiagen). Read mapping to the human reference genome GRCh37 and low frequency variant calling was performed using a customized workflow (described in more detail in Additional file 1) designed with the CLC Genomic Workbench, based on the “Identify QIAseq DNA Somatic Variants (Illumina)” workflow. For samples at or above 25% dd-cfDNA (as determined by ddPCR), the allele fraction for SNPs heterozygous in the recipient is expected to deviate significantly from 50%, and thus, the range of 25–75% used to identify such SNPs (Additional file 1) could not be used. In such samples, a matching plasma or urine sample from the same patient with a %dd-cfDNA below 20% was used, if available, or a recipient gDNA sample obtained from buffy coat was sequenced to identify SNPs homozygous in the recipient and calculate the %dd-cfDNA. The Variant Call Format (VCF) files generated using the described workflow were used to subsequently calculate the %dd-cfDNA and absolute number of dd-cfDNA copies as described in Additional file 1. Downstream data analysis was performed using Python v3.9.

The Python scikit-learn library v0.22.2 [17], implementing the expectation-maximization (EM) algorithm (n_components = 3), was used to categorize SNPs classified as homozygous in the recipient into three clusters based on their %dd-cfDNA and to calculate the mean %dd-cfDNA and weight by cluster. With these means and weights, the mean sample %dd-cfDNA using the informative SNPs was calculated (Eq. 2). A sample-specific noise factor, as the mean %dd-cfDNA calculated from the SNPs homozygous for the same allele in both the donor and recipient, was subtracted from the sample %dd-cfDNA.

Equation 2: Calculation of sample mean %dd-cfDNA using the EM algorithm results. The subscripts hetero and homo stand for heterozygous and homozygous. The subscript homo, donor stands for the SNPs homozygous in only the donor and homo, both stands for the SNPs that are homozygous in both the recipient and donor.

The mean absolute donor copies were calculated by averaging the donor copies of the SNPs in the homozygous group for the donor divided by two and the donor copies of the SNPs in the heterozygous SNP group.

2.6. Dd-cfDNA Quantification by AlloSeq® cfDNA

Using the AlloSeq® cfDNA (RUO) kit (CareDx, Brisbane, CA, USA), libraries of 24 cfDNA samples were prepared simultaneously with an input of 10 ng and sequenced on a MiSeq instrument (Illumina) employing v3 chemistry with 150 cycles according to the kit manufacturer’s manual (IFU084, v3.1, 10.2020). The FASTQ files were analyzed and the %dd-cfDNA was calculated using the AlloSeq® cfDNA Software (IFU085, v3.0, 07.2020). To avoid additional recipient genotyping, all samples analyzed with AlloSeq® cfDNA had a %dd-cfDNA below 20% as determined using ddPCR.

2.7. Statistical Analysis

All statistical analyses were performed with the open-source programming language R for statistical computing [18] or Python v3.9. Data visualization was done with the R package ggplot2 v3.3.5 [19] and the Python library matplotlib v3.4.3 [20]. For correlation analysis the squared Pearson correlation coefficient as R² and the Kendall’s Tau-a coefficient as τ were calculated. For regression analysis, the Passing Bablok and the linear least squares regression method were used. The 0.95% confidence interval (95%-CI) for the Passing Bablok regression were calculated with the bootstrap(quantile) method. To test for heteroscedasticity the Goldfeld-Quandt test was used, implemented in the R package lmtest v0.9-39 [21]. Bland-Altman, Q-Q and histogram plotting were performed using the R package blandr v0.5.1 [22]. Statistical testing between two continuous variables was performed with the Wilcoxon rank-sum test.

3.1. Validation

To assess the linearity of the QIAseq HTS method, a spike-in mixture series (n = 5) was measured using QIAseq with cfDNA from two unrelated healthy volunteers. Linear regression analysis indicated a strong linearity (R² = 0.999, p < 0.001; Fig. 1a) in the range of 0.1–1.5%. An additional spike-in dilution series (n = 6) measured with QIAseq HTS and ddPCR with two different assay combinations showed a strong linear correlation (R² > 0.999, p < 0.001; Fig. 1b) between both methods in the range of 0.5–40% without any systematic bias with an intercept at -0.41 (95%-CI: -1.03-0.16) but a small proportional bias (slope: 0.96; 95%-CI: 0.71–0.99). Furthermore, 20 plasma cfDNA samples from healthy volunteers were measured which showed a median %dd-cfDNA of 0.0075% (interquartile range, IQR): 0.000-0.037%).

For all samples measured in this study where the EM algorithm was applied, excluding one sample where the patient received an organ from his mother resulting in only heterozygous SNPs for the donor, the mean %dd-cfDNA was compared (n = 111) between the two groups of SNPs which were inferred to be heterozygous and homozygous, respectively, in the donor. A very similar %dd-cfDNA was observed in both groups (R² = 0.999, p < 0.001, Additional file 2, Figure S2). The median noise, calculated as the mean %dd-cfDNA from the SNPs inferred to be homozygous for the same allele in the donor and recipient, was not different from the noise observed in the healthy volunteer samples (p = 0.97) with 0.009% (IQR: 0.000-0.049%).

In the assessment of specificity of the HLA-DRB1 ddPCR assays, no unspecific amplification of nontarget alleles was observed in twelve homozygous carriers of different common alleles (DRB1*01:01, 03:01, 04:01, 04:04, 07:01, 08:01, 11:01, 11:04, 13:01, 13:02, 14:01, and 15:01). Additionally, the correlation between both assay combinations used to measure the total copies of a sample did not markedly differ whether both assay combinations measured the total copies directly (R² = 0.95, p < 0.001, n = 46), indirectly as the sum of donor and recipient copies (R² = 0.99, p < 0.001, n = 69) or one directly and the other indirectly (R² = 0.89, p < 0.001, n = 164) (Additional file 2: Figure S9).

3.2. Comparison between ddPCR and HTS (QIAseq)

To directly compare ddPCR to HTS (QIAseq), we assessed a total of 116 samples, of which 60 were collected early post-transplantation (< 1 month) while the remaining 56 were collected at routine follow-up appointments. Of those, eight early post-transplantation plasma samples were excluded because SNPs, which had a %dd-cfDNA of zero in urine were > 0% in plasma, suggesting the presence of third-party cfDNA, likely from a blood transfusion. An additional three plasma samples, all collected from the same patient post-transplantation, were excluded, as there was strong evidence that a blood transfusion introduced a substantial amount of third-party cfDNA, even though only plasma was available for analyses and the data could therefore not be compared to urine. The remaining 105 samples consisted of 38 plasma and 67 urine samples, of which 79 were collected from kidney, 24 from liver and two from a double transplant recipient (plasma and urine) who received both a kidney and liver from the same donor. Of those 105 samples, the donor cfDNA copies were indirectly measured with ddPCR (i.e. using subtraction of recipient copies from total copies; see Methods 2.4.3) in only two assay combinations and the recipient copies were indirectly determined in 84 assay combinations. Based on the ddPCR %dd-cfDNA (> 29%) of six urine samples, cellular gDNA from the recipient was additionally sequenced for the calculation of the %dd-cfDNA (see Methods 2.5.3). For 19 urine samples (> 18%) the matching plasma sample (< 20%) from the same patient was used, while for four plasma samples from liver transplant recipients the matching urine sample (< 8%) was used to identify SNPs with homozygous recipient genotype. Finally, for two post-transplantation samples, another sample from the same patient collected at a different time point with a %dd-cfDNA < 20% was used.

After comparing the proportion of SNPs with zero %dd-cfDNA among all SNPs homozygous in the recipient for a sample to the corresponding sample %dd-cfDNA (Additional file 2, Figure S12), we chose 1% as the threshold, below which a random sampling effect was observable, i.e. no donor-specific sequences were observed for some SNPs even if a donor-specific allele was present due to random sampling error. Consequently, below this threshold, the %dd-cfDNA calculation based on the EM algorithm was expected to produce biased results due to this sampling error. Therefore, in all samples with a %dd-cfDNA < 1%, the mean %dd-cfDNA over all homozygous recipient SNPs was considered, while above 1%, the %dd-cfDNA was calculated from the EM algorithm results.

The %dd-cfDNA showed a strong correlation (R² = 0.977, p < 0.001, Fig. 2a) between both methods with a slight bias towards a higher percentage measured with the HTS method and a significant heteroscedasticity (p < 0.001). Bland-Altmann plotting confirmed the bias (Additional file 2, Figure S6a); however, it was not significant below a %dd-cfDNA of 5%. The correlation was not markedly different when measurements were differentiated by body fluid (urine: R² = 0.988; plasma: R² = 0.969), whereas the proportional bias was only present for urine and not for plasma (Table 1).

For the same samples, we also compared the absolute number of dd-cfDNA copies between both methods. For this correlation analysis, we excluded six samples with > 20’000 dd-cfDNA copies to reduce the influence of strong outliers on the analyses. A strong correlation was observed (τ = 0.779, p < 0.001, R² = 0.863, Fig. 3). There was no systematic bias (intercept: -0.058; 95%-CI: -1.33-0.269), however, a much lower level of donor copies was measured with the QIAseq method (slope: 0.25; 95%-CI: 0.19–0.26).

Table 1

Passing Bablok regression parameters of the different method comparisons for the %dd-cfDNA and for specific sample subsets.
		intercept			slope
		estimate	95%-CI		estimate	95%-CI
QIAseq versus ddPCR	overall	0.005	-0.032	0.109	1.11	1.05	1.15
	urine (n = 67)	0.147	0.000	0.783	1.12	1.04	1.16
	plasma (n = 38)	-0.018	-0.058	0.072	1.04	1.00	1.12
	follow-up (n = 56)	-0.016	-0.057	0.006	1.11	1.03	1.15
	post-transplantation (n = 49)	0.414	-0.020	0.805	1.08	1.02	1.18
QIAseq versus AlloSeq cfDNA		-0.098	-0.165	0.001	1.05	1.01	1.11
AlloSeq cfDNA versus ddPCR		-0.093	-0.196	0.112	0.99	0.81	1.05
The 95% confidence interval was calculated with the bootstrap(quantile) method.

3.3. Comparison to commercial kit

We further compared the QIAseq method with the commercially available AlloSeq® cfDNA kit (CareDx) in a subset of 38 samples (13 urine and 25 plasma). We could show that both HTS methods strongly correlated (R² = 0.993, p < 0.001, Fig. 2b) without any systemic bias, only minimal proportional bias (Table 1) and no significant overall bias between both methods (Bland-Altman plot, Additional file 2, Figure S6b). Similarly, for a subset of 31 samples measured both with AlloSeq® cfDNA and ddPCR, there was a strong correlation (R² = 0.909, p < 0.001, Fig. 2c) without any bias.

3.4. Dd-cfDNA levels in Stable Allograft Recipients

Among a total of 30 urine samples analyzed by ddPCR from stable kidney recipients (Table 2) as supported by a negative biopsy result or low average serum creatinine, the median %dd-cfDNA was 39.5% (IQR: 21.8–58.5%) ranging from 0.54–81% (Fig. 4b). The median dd-cfDNA copies/ml urine was 239 (IQR: 135–506) (Fig. 4d) and 483 (IQR: 152–988) for recipient copies/ml. Urine creatinine levels at the time (± 1.5 h) of urine collection were available for only 21 samples. When normalized by UCr [µmol/ml], the median donor copy number per µmol UCr was 36.6 (IQR: 18.4–109) (Fig. 4e). The median recipient copy number was 80.2 (IQR: 24.3–620) copies/µmol UCr. A median of 2.6 ng cfDNA per ml (IQR: 1.5–5.6 ng/ml) were extracted from a median urine volume of 39.5 ml (IQR: 21.8–45.3 ml). For the stable kidney recipient urine samples with creatinine levels, the median total cfDNA concentration was 0.39 pg (IQR: 0.18-2 pg) per µmol UCr.

In plasma of the same 30 stable kidney recipients, the median %dd-cfDNA was 0.19% (IQR: 0.01-0.43%) with a maximum of 0.73% (Figure 4a). The median dd-cfDNA copy number/ml plasma was at 5.0 (IQR: 1.8-12.9) (Figure 4c) and 2320 (IQR: 1733-3347) for the recipient copies/ml. For the stable recipient plasma samples, a median of 13.0 ng cfDNA per ml (IQR: 8.2-17.5 ng/ml) was extracted from 4.0 ml (IQR: 3.5-4.2 ml). One outlier with increased levels of donor copies/ml compared to the other patients could each be observed in plasma and urine. However, the samples with increased levels in urine and plasma respectively, did not originate from the same recipient.

For the stable kidney transplant recipients, the interquartile range of the copies/ml plasma and copies/µmol UCr relative to the median was calculated. In plasma, this ratio was 3.2-fold greater for the dd-cfDNA copies compared to the recipient copies. In urine, the opposite was true with a factor three greater ratio for the recipient copies compared to the dd-cfDNA copies. The ratio for the dd-cfDNA was similar in plasma (2.22) and urine (2.48) but different for the recipient copies (plasma: 0.70; urine: 7.43).

In addition, urine and plasma of eight stable liver recipients (Table 2) were analyzed by ddPCR. With a median of 2.2% (IQR: 0.72–4.1%) (Fig. 5a) in plasma, the %dd-cfDNA was higher than in plasma from stable kidney recipients (p < 0.001). The number of dd-cfDNA copies/ml was 120 (IQR: 85.0-138) (Fig. 5c) and 4918 (IQR: 2165–14434) for the recipient-derived cfDNA. A median of 25.2 ng cfDNA per ml (IQR: 10.8–41.5) was extracted from 4.3 ml (IQR: 4.0-4.6) of plasma.

Table 2

Stable solid organ transplant patient demographics
Recipient Characteristics	Kidney (n = 30)	Liver (n = 8)
Age, years
mean (SD)	57 (13)	56 (16)
median	59	63
range	30–77	31–71
Gender
male (%)	23 (76.7)	6 (75.0)
female (%)	7 (23.3)	2 (25.0)
Time post-transplant, days
mean	1716	1289
median	939	355
DSA status
DSA positive (%)	7 (23.3)	4 (50.0)
DSA negative (%)	23 (76.7)	3 (37.5)
unknown (%)		1 (12.5)
Indication for transplantation
alcohol-related (%)		3 (37.5)
virus-related		1 (12.5)
PSC		2 (25.0)
glomerulonephritis	5 (16.7)
cardiovascular	5 (16.7)
ADPKD	8 (26.7)
post SOT-CKD	1 (3.33)
other	11 (36.7)	2 (25.0)
DSA: donor-specific antibodies; PSC: primary sclerosis cholangitis; ADPKD: autosomal dominant polycystic kidney disease; post SOT-CKD: post solid organ transplantation chronic kidney disease

In urine of liver recipients on the other hand, almost no dd-cfDNA copies could be detected with a median of 0.016 copies/ml and a maximum of 0.378 copies/ml (Fig. 5d). The median of recipient-derived cfDNA copies was 1737 copies/ml (IQR: 928–3888). The %dd-cfDNA was measured at 0.0007% with a maximum at 0.0378% (Fig. 5b), which was significantly lower (p < 0.001) compared to the kidney transplant patients. From urine, a median of 6.5 ng cfDNA per ml (IQR: 3.2–13.3) were extracted from 40.0ml (IQR: 40.0-46.2).

There was a significantly higher (p < 0.05) concentration of total cfDNA in plasma from liver transplant recipients when compared to stable kidney recipients while the difference was not significant in urine.

In all 38 samples from stable solid organ transplant recipients, the donor copies were measured directly while for 66 assay combinations, the recipient copies were measured indirectly (see Methods 2.4.3).

As for a set of patients %dd-cfDNA (n = 127) and absolute copies (n = 36) were measured in both urine and plasma, we assessed whether any correlation could be detected between the two body fluids. However, no significant correlation could be detected between urine and plasma for relative nor for absolute levels in both stable and early post-transplantation patients (Fig. 6a/b).

With the emergence of dd-cfDNA as a novel minimally invasive biomarker for allograft rejection supported by numerous clinical validation studies, a variety of analytical methods for dd-cfDNA quantification have been developed and utilized. However, besides the analytic validation, studies directly comparing different quantification approaches are lacking. Here we showed that ddPCR and HTS, the two most commonly used methods for dd-cfDNA quantification, are highly comparable and display only minimal bias for %dd-cfDNA. Moreover, this study represents to our knowledge the first development of an HTS method using UMI to enable absolute quantification of dd-cfDNA copies. Finally, urine was shown to contain substantial amounts of dd-cfDNA in stable kidney allograft recipients, albeit with a highly variable fractional abundance, supporting the requirement for absolute quantification to unlock the potential of urinary-derived dd-cfDNA as a fully non-invasive biomarker for renal graft transplant surveillance.

This HTS method incorporating UMIs for dd-cfDNA quantification without the need for donor-genotyping below 25% showed a high agreement of the %dd-cfDNA with ddPCR using assays targeting HLA-DRB1 alleles. The range of %dd-cfDNA measured in this study was between 0% and > 90% and included values greater than the range relevant in clinical diagnostics for plasma-based analyses, especially in hepatic and renal graft transplantation beyond the first month post-transplantation. However, as abnormal dd-cfDNA kinetics in the early post-transplantation phase with potentially high %dd-cfDNA of above 25% might be indicative of early adverse events in kidney transplant recipients such as urinary tract infections, pre-renal acute kidney injury or surgical complications among others [23], the accordance of both methods in a broad range is of great importance. Moreover, in the case of allogeneic hematopoietic cell transplantation, dd-cfDNA makes up the vast majority of cfDNA fragments [24] and therefore the relevant range is the opposite starting at 100%. Important to highlight is that these results show that even though both methods are based on completely different technologies and have different molecular targets, they result in a similar %dd-cfDNA. cfDNA release has been shown to be highly dependent on histone packaging and its susceptibility to DNase degradation, while also correlating with the transcription level of genes [25]. As HTS methods interrogate multiple SNPs in the genome, any differences in the frequency of SNPs among the pool of (dd-)cfDNA fragments due to different cfDNA fragmentation would likely be canceled out. For ddPCR, on the other hand, with the assays used here targeting only a single locus in the HLA-DRB1 gene for the donor and recipient, such an effect is not expected. Moreover, even though the novel HTS method using UMI has not been as extensively validated as other HTS methods, we showed that a similar %dd-cfDNA could be measured when compared with AlloSeq® cfDNA, an analytically and clinically validated HTS assay. It is also worth mentioning that even though AlloSeq® cfDNA was validated for dd-cfDNA from plasma only, we also included urine samples in our analyses and still observed a high agreement between both methods. In contrast to the AlloSeq® cfDNA bioinformatic analysis, which corrected for close donor-recipient relatedness using constants, we implemented an EM algorithm that adjusts automatically for any skewing in the proportion of different donor SNP genotypes among recipient homozygous SNPs. The strong concordance of the %dd-cfDNA from both donor genotypes among informative SNPs corroborates the robustness of the algorithm. The algorithm can also be applied for a parent-child donor relationship with only heterozygous informative SNPs by defining the number of clusters as two instead of three.

To our knowledge, this is the first published incorporation of UMI to enable a more accurate absolute quantification of dd-cfDNA using HTS. The strong correlation with ddPCR suggests that HTS with UMI can be used to further evaluate the feasibility of HTS-based absolute dd-cfDNA quantification for minimally or non-invasive graft monitoring using dd-cfDNA. While the correlation with ddPCR was strong, a substantial proportional bias towards a lower number of dd-cfDNA copies detected by the HTS method was observed. This bias indicates that a lower proportion of input cfDNA molecules was sequenced with HTS compared to ddPCR based quantification, which could have different causes. First, there could have been a significant loss of fragments during ligation of adapters with the UMIs. Further, bead-based clean-up after adapter ligation before target enrichment might have substantially contributed to the loss of fragments. To utilize a method-independent diagnostic cutoff for absolute dd-cfDNA copy numbers, it is necessary to evaluate whether a sample-independent correction factor could be used to adjust absolute dd-cfDNA quantities from different methods. The use of such a correction factor could be feasible given that the ligation efficiency and fragment loss during clean-up are expected to be similar for all samples under the same conditions. Furthermore, the library preparation protocol has already been further improved by the manufacturer, replacing bead-based with enzymatic clean-up steps with the potential to minimize UMI loss after ligation and improving UMI ligation itself, which could increase the number of dd-cfDNA fragments in the library while potentially also minimizing variability.

We further applied ddPCR to determine and compare absolute and relative levels of dd-cfDNA in plasma and urine from stable liver and kidney transplant recipients. Plasma is the best-established source material for dd-cfDNA quantification so far and compared to urine, there is extensive literature on dd-cfDNA levels in plasma from allograft recipients. In 2017, the DART cohort-based study conducted by Bromberg et al. [26] investigated the median %dd-cfDNA and its variation in a reference kidney transplant population with the validated assay from CareDx. The median %dd-cfDNA of 0.19% (IQR: 0.01–0.43%) observed in this study is in accordance with the median of 0.21% (IQR: 0.12%-0.39%) in 380 blood samples reported by Bromberg et al. Similarly, Oellerich et al. [7] and Bloom et al. [4] reported median values of 0.29% (IQR: 0.17%-0.56%) and 0.30% (IQR: 0.14%-0.77%), respectively. For hepatic allograft recipients, Schütz et al. [3] conducted one of the most comprehensive studies in terms of the number of recipients included (n = 115). With a median %dd-cfDNA of 3.3% (95% CI 2.9%-3.7%) among stable liver transplant recipients, the level is similar even though a bit higher than the 2.2% (IQR: 0.72–4.1%) found in this study.

There is, however, limited data on the level of absolute copies in plasma from solid organ transplant recipients. The median number of dd-cfDNA copies/ml in kidney recipients with a negative biopsy in the cohort from Whitlam et al. [8] was 7 (IQR: 5–11), which was similar to the 5 copies/ml (IQR: 2–13) observed here. Oellerich and his colleagues [7] also investigated the absolute levels in stable kidney transplant recipients and found a median of 25 copies/mL (IQR: 11–60), which was five times higher than the number of dd-cfDNA copies detected here. One of the more prominent reasons for this discrepancy could be that both in our study as in that from Whitlam et al. no correction for the cfDNA extraction efficiency was used, while Oellerich et al. used an artificial spike-in to adjust for the extraction efficiency. Nevertheless, despite this difference in the mean dd-cfDNA copies observed, the inter-patient variation, expressed as the interquartile range relative to the median, was similar with 2.2 in our patient cohort and 2.0 in the study by Oellerich et al. Goh and colleagues [27] were to our knowledge the only ones who investigated absolute levels of dd-cfDNA in plasma from stable liver recipients and found a median of 66 copies/ml (IQR: 50–105) at day 42 post-transplantation in patients (n = 14) with an uneventful recovery. This is approximately half the dd-cfDNA copies/ml found here. However, it is noteworthy that the plasma samples from over half the stable liver recipients included here were collected one year post-transplantation and can therefore not be directly compared to the dd-cfDNA values at day 42. Further, Goh et al. may have underestimated the true number of copies as they did not adjust for non-amplifiable copies due to the amplicon length of the assays (50–130 bp) [28].

In contrast to plasma, urine would be a truly non-invasive source material with the additional benefit that it could be collected at home by the patients, and that it can easily be obtained in larger quantities compared to plasma. For this reason, we investigated the levels of dd-cfDNA in urine from solid organ recipients and found that the median %dd-cfDNA of 39.5% (IQR: 21.8–58.5%) in urine from stable kidney transplant recipients was slightly lower than the results revealed in other investigations such as from Lee et al. [29] with a mean of 53.3% (IQR: 21–92%) (n = 8) analyzed by sex-mismatch ddPCR and Burnham et al. [30] with a mean of 51.4% (n = 4) analyzed by HTS. With 30 samples from stable kidney recipients investigated here, the sample size was substantially larger in this study, which may explain the slight discrepancy to the two previous investigations based only on small patient numbers.

The only known study investigating urinary dd-cfDNA absolute copy numbers normalized by urinary creatinine levels so far was carried out by Sigdel et al. [31], reporting a mean number of 2.67 copies/µg creatinine. Based on a molar mass for creatinine of 113.12 gmol^− 1, this corresponds to about 0.02 copies/µmol UCr, which is substantially lower than the 36.6 copies/µmol UCr measured in this study. A major reason for this discrepancy is that Sigdel et al. [31] log₂ transformed the copies/ml before normalizing them by the urine creatinine levels. Applying the same transformation to the data in our study, the difference to the results from Sigdel et al. was strongly reduced with 5.2 copies/µmol UCr but remained substantial. A possible reason to explain the remaining difference might be that another method with a different extraction efficiency was used to extract the urinary cfDNA.

The results of the % dd-cfDNA in urine from stable kidney recipients revealed a large variability with values ranging from 0–80%, suggesting that urinary %dd-cfDNA is likely not a good biomarker candidate to identify or exclude kidney allograft rejections. This is supported by the observation that the extensive variation of the %ddcfDNA can predominantly be explained by the substantial fluctuations of recipient copies and less so by the donor copy variability. This large variation in recipient copies may potentially mask relevant changes in the amount of dd-cfDNA in urine if expressed relative to the total copies. Considering these results, the quantification of absolute dd-cfDNA levels will likely be needed to further evaluate the potential of urinary dd-cfDNA for allograft monitoring.

With the comparison of urine and plasma for the same stable allograft recipients, it was observed that for patients who received a kidney, both the %dd-cfDNA and absolute copies were higher in urine compared to plasma. One might thus hypothesize that more DNA is released from the kidney into the urine than into the blood circulation. A difference in cfDNA release dynamics is also supported by the lack of correlation of relative and absolute levels of dd-cfDNA between plasma and urine from the same patients. However, before any conclusions can be drawn, the comparison of the total amount of cfDNA released into the blood and urine would be needed to confirm this.

With our analysis of urine samples from stable liver transplant recipients, we aimed to gain further understanding about the phenomenon of transrenal dd-cfDNA, i.e. dd-cfDNA passing through the kidney into the urine. Even though a substantial number of dd-cfDNA copies could be detected in plasma in all liver recipients, interestingly, virtually no copies from the donor organ could be detected in urine from the same patients. These findings thus suggest very low levels of transrenal cfDNA in stable liver recipients. To our knowledge, there are no studies investigating relative nor absolute levels of transrenal dd-cfDNA originating from hepatic transplants. One study has investigated cfDNA in urine using methylation patterns specific for hepatocytes in healthy humans. Cheng et al. [32] detected virtually no transrenal cfDNA from hepatocytes, although no exact values were reported. Liver-derived cfDNA with just over 1% [33] made up the third-largest contributor to circulating cfDNA in plasma from healthy individuals besides blood- and vascular endothelium-derived cells. Based on this study and on our results, it is therefore unlikely that dd-cfDNA in urine could be used to monitor non-renal organ allografts with the methods currently available.

Of note, however, the extraction kit used for urinary-derived cfDNA had an isolation size range of 100 − 23’000 bp. Markus et al. [34] showed that the modal fragment size was 81 bp for all cfDNA fragments found in urine while the fragment size was 167 bp in plasma. It can therefore not be excluded that transrenal cfDNA predominantly consists of fragments significantly below 100 bp length. If transrenal cfDNA indeed has a shorter fragment length compared to plasma cfDNA, such fragments may have been lost during cfDNA isolation in our study. Biophysical studies on the length of transrenal dd-cfDNA in solid organ transplant recipients are lacking and results of studies on the length of transrenal cfDNA in other pathologies or pregnancy should not be directly translated, therefore the length of these fragments is still unknown. Alternative extraction kits or customized protocols should be investigated for the potential to extract short fragments so transrenal cfDNA might potentially be more efficiently isolated in solid organ transplant recipients. Comparison of kits with different degrees of short fragment extraction efficiencies would also reveal the extent of the effect that the extraction method has on the detection of transrenal DNA in urine.

Finally, this study has some limitations. First, a bit less than half of the 105 samples used for the ddPCR to HTS comparison were plasma samples. Therefore, body fluid-specific method agreement results are of limited statistical power. However, as both combined showed good agreement, big deviations are not expected if more samples were to be measured for each of the body fluids. A further limitation are the criteria, on which patients were deemed in a stable phase. For some kidney transplant recipients, a biopsy was not available and the decision was based on a low average serum creatinine, which has been shown to be an unsatisfactory biomarker to detect subclinical rejection. Similarly, the decision for the liver transplant patients was based on the levels of liver enzymes and total bilirubin, which show a low sensitivity and specificity in detecting subclinical acute cellular rejection. Since no liver biopsies were performed, no histological correlations could be shown. Therefore, it cannot be excluded that the data shown here also included patients with a subclinical rejection, which is associated with increased absolute and relative dd-cfDNA.

Overall, this first head-to-head comparison of the most commonly used approaches for the relative quantification of dd-cfDNA yielded highly comparable results with no or minimal biases. With the first incorporation of UMI in a dd-cfDNA HTS assay for dd-cfDNA quantification, we demonstrated the suitability of the presented custom HTS method for absolute dd-cfDNA quantification, even though the definition of a method-independent diagnostic cutoff for absolute dd-cfDNA may not be feasible given the substantially lower levels observed by HTS compared to ddPCR. Finally, high variability in recipient-derived cfDNA in urine indicates that methods enabling absolute dd-cfDNA quantification may be required to further evaluate urinary-derived dd-cfDNA for renal allograft surveillance.

dd-cfDNA

donor-derived cell-free DNA

ddPCR

droplet digital PCR

HTS

high-throughput sequencing

SNP

single-nucleotide polymorphisms

expectation-maximization

UCr

urinary creatinine

UMI

unique molecular identifiers

Ethics approval and consent to participate

All patients have signed a written consent and the healthy volunteers were anonymized. The study was approved by the ethics committee of the Canton of Bern, Switzerland (2019-00730).

Consent for publication

Not Applicable

Availability of data and materials

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This study has been funded by the Swiss National Science Foundation grant number 310030_188762, the Hemmi foundation and the Fondation Johanna Dürmüller-Bol.

Authors' contributions

UA and DS designed the study; DS, VB, CK recruited patients; NK and SA isolated and analyzed the cfDNA from samples; SA designed and validated the HTS assay with NK; NK performed the bioinformatic, data and statistical analysis; FS collected and summarized the clinical data; CL provided scientific advice; NK and UA drafted the manuscript; UA supervised the study, contributed to results interpretation and statistical analysis. All authors read, reviewed and approved the final manuscript.

Acknowledgments

The authors would like to thank all the patients and their families, and the healthy volunteers for participation. We further thank all the University Institute of Clinical Chemistry, Clinical Genomics Lab, Human Genetics department technicians and staff for their laboratory support, namely Daniel Schärer, Ain Alexeeva, Gisela Andrey, Anna Kopps, Janic Teutsch, Daniela Dumke and Philippe Reuge. We also thank Katharina Staufer for her initial support in the recruitment of liver recipients, and all nurses and medical staff involved in sample collection. Lastly, we are also grateful to the representatives of Qiagen and CareDx Inc. for their support and insights.

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Additionalfile1subm.pdf
All additional method descriptions necessary for reproducing the research presented in this manuscript
Additionalfile2subm.pdf
All additional figures not presented in the main text
Additionalfile3subm.xlsx
A list of the SNPs included in the QIAseq panel

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Comparison of Methods for Donor-Derived Cell-Free DNA Quantification in Plasma and Urine from Solid Organ Transplant Recipients

Status:

Version 1

Abstract

Background

Methods

Results

Conclusions

Figures

1. Introduction

2. Methods

2.1. Patient Recruitment and Sample Collection

2.2. cfDNA and gDNA Extraction

2.3. Spike-in cfDNA Dilution Series

2.4. Dd-cfDNA Quantification by ddPCR

2.4.1. Assays and Validation

2.4.2. Experimental Setup and Measurement

2.4.3. ddPCR Data Analysis

2.4.4. Adjusting for Non-Amplifiable Fragments and Concentration of Urine

2.5. Dd-cfDNA Quantification by QIAseq HTS

2.5.1. Assay Design and Validation

2.5.2. Library Preparation and Sequencing

2.5.3. Bioinformatic Analysis

2.6. Dd-cfDNA Quantification by AlloSeq® cfDNA

2.7. Statistical Analysis

3. Results

3.1. Validation

3.2. Comparison between ddPCR and HTS (QIAseq)

3.3. Comparison to commercial kit

3.4. Dd-cfDNA levels in Stable Allograft Recipients

4. Discussion

5. Conclusion

6. List of Abbreviations

7. Declarations

8. References

Supplementary Files

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