dd-cfDNA Research Articles

Local laboratory-run donor-derived cell-free DNA assay for rejection surveillance in heart transplantation-first six months of clinical experience.

Donor-derived cell-free DNA (dd-cfDNA) is a blood biomarker detecting graft injury with high negative predictive value. While non-invasive strategies for heart transplant (HTx) rejection surveillance are widely adopted in the United States with centralized testing, data on the feasibility of dd-cfDNA assay at the local level are lacking. Here, we report the first 6 months of experience with a local laboratory-run dd-cfDNA assay in the routine clinical surveillance setting. Twenty-six HTx patients with stable graft function were transitioned from endomyocardial biopsy-based (EMB) to dd-cfDNA-led rejection surveillance using a commercially available next-generation sequencing-based assay. In the 90 samples analyzed, dd-cfDNA fraction remained continuously low in most patients, thus 88% of surveillance EMBs could be safely avoided. In the case of ≥.25% dd-cfDNA, EMB was performed. There was no missed rejection. Our data show the feasibility to analyze dd-cfDNA at the local level and successful implementation of this non-invasive surveillance method into clinical practice, thus considerably reducing the frequency of invasive surveillance EMBs.

Biomarkers in the Management of the Lung Transplant Allograft: A Focus on Donor-derived Cell-free DNA

Advances in the use of molecular biomarkers to guide management of lung transplant recipients are emerging. One such biomarker, donor-derived cell-free DNA (dd-cfDNA), has the potential to alter traditional approaches of lung transplant management, including the detection of acute rejection or infection, surveillance monitoring strategies, risk stratification of vulnerable patient populations and molecular phenotyping of various forms of allograft injury. The emergence of novel genetic and epigenetic approaches utilizing cfDNA provides further promise for the elucidation of pathophysiological mechanisms and treatment targets in various disease states. This review aims to employ the current state of the evidence to cover the biological basis, clinical applications and future directions for the use of dd-cfDNA in lung transplant recipients.

#3743 MULTIDIMENSIONAL RISK ASSESSMENT OF KIDNEY ALLOGRAFT REJECTION USING DONOR DERIVED CELL-FREE DNA

Abstract Background and Aims Post-transplantation patient care requires development and validation of non-invasive biomarkers to improve allograft monitoring and prevention from unnecessary and costly biopsies. Reports have suggested the association of donor derived cell-free DNA (dd-cfDNA) with allograft rejection. However, there is no proof of its added value on standard of care in large, unselected and deep phenotyped cohorts. Method We enrolled 1134 kidney transplant recipients having concomitant evaluation of allograft histology, anti-HLA DSA and functional parameters between April 2013 and June 2018 in the derivation cohort, representing 1415 biopsies. Dd-cfDNA was measured in plasma at the time of the biopsy. Diagnoses were performed using Banff 2019 criteria. 171 AMR, 34 TCMR and 17 mixed rejections occurred. Parameters associated with rejection were assessed using uni- and multivariable logistic regression. We then developed a risk model using the variables that were independently associated with kidney rejection. The validation cohort comprised 1929 evaluations including 499 evaluations in one Belgian center and 1430 evaluations in nine North American centers. Results Higher levels of dd-cfDNA were observed for AMR and TCMR or both compared to other diagnoses (Fig. 1A). Dd-cfDNA incrementally increased with Banff acute lesions without significant increase for chronic lesions. In multivariable analysis, the variables independently associated with rejection were anti-HLA DSA (P<0.0001), dd-cfDNA (P<0.0001), eGFR (P<0.033), proteinuria (P = 0.016), and previous history of rejection (P<0.0001). Dd-cfDNA remained independently associated with kidney allograft rejection in validation cohorts from Belgium (P = 0.0006) and North America (P<0.0001). Discrimination of the model without dd-cfDNA was 0.777 and 0.821 with its inclusion, showing its added value (Fig. 1B). The good discrimination performances of the model with dd-cfDNA were also confirmed in the validation cohorts from Belgium (AUC: 0.815) and North America (AUC: 0.826). Conclusion We here demonstrate the independent and added value of dd-cfDNA in addition to conventional features to predict rejection. This first integrative system shows improved performance for patient monitoring and could help physicians in decision-making process.

#4743 OMNIGRAF (COMBINED GENE EXPRESSION AND DD-CF DNA) USE IN MONITORING RENAL ALLOGRAFT REJECTION IN MULTIORGAN TRANSPLANT RECIPIENTS

Abstract Background and Aims To date there is no clinically available non-invasive biomarker to monitor for renal allograft rejection in multiorgan transplant recipients. TruGraf (Gene expression profile) and TRAC (dd-cf DNA), as part of the biomarker panel OmniGraf, have shown to be reliable “rule out” tests for subclinical rejection in stable kidney transplant recipients. However, the validity and utility of these tests in monitoring for renal allograft rejections in the setting of multiorgan transplants has yet to be assessed. The aim of this study is to assess the feasibility of using the biomarkers individually, as well as together in the OmniGraf biomarker panel, to assist in making the decision to perform a renal allograft biopsy in multiorgan transplant recipients. Method This is an ongoing prospective, observational sample collection study in any multiorgan (kidney + non-renal organ) undergoing a renal allograft biopsy. Biopsies were considered positive if they showed rejection in the kidney based on Banff 2019 and included borderline changes as positive results. Copper-Pearson 95% CI were used for sensitivity, specificity, and accuracy; standard logit CIs were used for NPV and PPV. For TruGraf, (TX = negative result) and (not-TX = positive test). For TRAC, 0.7% was used as the threshold for all organs except for those that had a liver transplant, where a threshold of 15% was used to determine positive vs. negative. Results Paired renal allograft surveillance biopsies and biomarker samples were available for 20 patients. Of the 20, 9 (45%) were liver-kidney transplants, 6 (30%) were heart-kidney transplants, and 5 (25%) were kidney-pancreas transplants. Nine (45%) of the patients were male, 14 (70%) were White, 5 (25%) were Black, and 1 patient was Hispanic. The mean age was 50 ± 13 years. Average SrCr was 1.3 ± 0.5 mg/dL. Of the 20 paired biopsies and samples, 2 (10%) had only TruGraf results, while the other 18 (90%) had OmniGraf (TruGraf and TRAC). There were 2 rejections (borderline changes). Diagnostic performance is presented in tables 1–4. Both TruGraf and TRAC individually had NPV of 89% and 85%, respectively, and PPV of 65% and 73%, respectively. When analyzing the performance of the OmniGraf biomarker panel, OmniGraf +/+ showed a 100% PPV and OmniGraf -/- showed a 100% NPV. Conclusion In this analysis of renal allograft biopsies in a multiorgan transplant cohort, TruGraf, TRAC (when using organ-dependent thresholds) had reasonable diagnostic performance as a “rule out” test. When the tests are combined into OmniGraf, the PPV and NPV were 100% when both tests agreed with each other. These outcomes justify potential use and the need for continued analysis of the utility of the OmniGraf biomarker panel in monitoring renal allograft in multiorgan transplant recipients.

All That Glitters in cfDNA Analysis Is Not Gold or Its Utility Is Completely Established Due to Graft Damage: A Critical Review in the Field of Transplantation.

In kidney transplantation, a biopsy is currently the gold standard for monitoring the transplanted organ. However, this is far from an ideal screening method given its invasive nature and the discomfort it can cause the patient. Large-scale studies in renal transplantation show that approximately 1% of biopsies generate major complications, with a risk of macroscopic hematuria greater than 3.5%. It would not be until 2011 that a method to detect donor-derived cell-free DNA (dd-cfDNA) employing digital PCR was devised based on analyzing the differences in SNPs between the donor and recipient. In addition, since the initial validation studies were carried out at the specific moments in which rejection was suspected, there is still not a good understanding of how dd-cfDNA levels naturally evolve post-transplant. In addition, various factors, both in the recipient and the donor, can influence dd-cfDNA levels and cause increases in the levels of dd-cfDNA themselves without suspicion of rejection. All that glitters in this technology is not gold; therefore, in this article, we discuss the current state of clinical studies, the benefits, and disadvantages.

Anything But a Biopsy: The Quest for Noninvasive Alternatives in Heart Transplantation.

Anything But a Biopsy: The Quest for Noninvasive Alternatives in Heart Transplantation.

An Initial Analysis of the Baseline Levels of Donor Derived-Cell Free DNA(Dd-Cfdna) After Pancreas Transplantation: A Prospective Study from High-Volume Centers in the US

Yoo, Ashley; Riedel, Alexandria BA; Qian, Ian BS; Bartosic, Amanda BS, MBA; Kibria, Gulam MPH, PhD; Abrams, Peter L MD, FACS; Odorico, Jon S MD; Cooper, Matthew MD, FACS; Bromberg, Jonathan S MD, FACS; Scalea, Joseph R MD Author Information

Donor-Derived Cell-Free DNA for Kidney Allograft Surveillance after Conversion to Belatacept: Prospective Pilot Study

Donor-derived cell-free DNA (dd-cfDNA) is used as a biomarker for detection of antibody-mediated rejection (ABMR) and other forms of graft injury. Another potential indication is guidance of immunosuppressive therapy when no therapeutic drug monitoring is available. In such situations, detection of patients with overt or subclinical graft injury is important to personalize immunosuppression. We prospectively measured dd-cfDNA in 22 kidney transplant recipients (KTR) over a period of 6 months after conversion to belatacept for clinical indication and assessed routine clinical parameters. Patient and graft survival was 100% after 6 months, and eGFR remained stable (28.7 vs. 31.1 mL/min/1.73 m2, p = 0.60). Out of 22 patients, 2 (9%) developed biopsy-proven rejection-one episode of low-grade TCMR IA and one episode of caABMR. While both episodes were detected by increase in creatinine, the caABMR episode led to increase in absolute dd-cfDNA (168 copies/mL) above the cut-off of 50 copies/mL, while the TCMR episode did show slightly increased relative dd-cfDNA (0.85%) despite normal absolute dd-cfDNA (22 copies/mL). Dd-cfDNA did not differ before and after conversion in a subgroup of 12 KTR with previous calcineurin inhibitor therapy and no rejection (12.5 vs. 25.3 copies/mL, p = 0.34). In this subgroup, 3/12 (25%) patients showed increase of absolute dd-cfDNA above the prespecified cut-off (50 copies/mL) despite improving eGFR. Increase in dd-cfDNA after conversion to belatacept is common and could point towards subclinical allograft injury. To detect subclinical TCMR changes without vascular lesions, additional biomarkers or urinary dd-cfDNA should complement plasma dd-cfDNA. Resolving CNI toxicity is unlikely to be detected by decreased dd-cfDNA levels. In summary, the sole determination of dd-cfDNA has limited utility in the guidance of patients after late conversion to belatacept. Further studies should focus on patients undergoing early conversion and include protocol biopsies at least for patients with increased dd-cfDNA.

An Initial Analysis of the Baseline Levels of Dd-cfDNA After Pancreas Transplantation: A Prospective Study From High-volume Centers in the United States.

One hundred eighty-one dd-cfDNA samples (n) were collected from 77 patients (N) up to 132 mo posttransplant. The median dd-cfDNA level among all subjects was 0.28% (0.13%, 0.71%). In simultaneous pancreas-kidney (SPK) transplant recipients, the median dd-cfDNA level was 0.29% (0.13%, 0.71%), and it was 0.23% (0.08%, 0.71%) in pancreas transplant alone (PTA) recipients. When isolating for when without infection or rejection, the median dd-cfDNA level was 0.28% (0.13%, 0.64%) for SPK and 0.20% (0.00%, 0.32%) for PTA. Both transplant types approached 1.0% ≤1 mo posttransplant followed by a decrease in median dd-cfDNA. During episodes of rejection or infection, median dd-cfDNA levels were greater among all transplant types. The mean dd-cfDNA level for all pancreas transplant recipients is <1.0%, consistent with the published kidney transplant rejection threshold (>1.0%), regardless of SPK or PTA. Early posttransplant dd-cfDNA levels are transiently higher than later measurements. Dd-cfDNA elevation also correlates with rejection and infection and thus is a promising biomarker for surveilling pancreas transplant dysfunction.

Droplet digital PCR-based testing for donor-derived cell-free DNA in transplanted patients as noninvasive marker of allograft health: Methodological aspects.

In solid organ transplantation, donor-derived cell-free DNA (dd-cfDNA) is a promising universal noninvasive biomarker for allograft health, where high levels of dd-cfDNA indicate organ damage. Using Droplet Digital PCR (ddPCR), we aimed to develop an assay setup for monitoring organ health. We aimed to identify the least distinguishable percentage-point increase in the fraction of minute amounts of cfDNA in a large cfDNA background by using assays targeting single nucleotide polymorphisms (SNPs). We mimicked a clinical sample from a recipient in a number of spike-in experiments, where cfDNA from healthy volunteers were mixed. A total of 40 assays were tested and approved by qPCR and ddPCR. Limit of detection (LOD) was demonstrated to be approximately 3 copies per reaction, observed at a fraction of 0.002%, and which would equal 6 copies per mL plasma. Limit of quantification (LOQ) was 35 copies per reaction, estimated to 0.038%. The lowest detectable increase in percentage point of dd-cfDNA was approximately 0.04%. Our results demonstrated that ddPCR has great sensitivity, high precision, and exceptional ability to quantify low levels of cfDNA. The ability to distinguish small differences in mimicking dd-cfDNA was far beyond the desired capability. While these methodological data are promising, further prospective studies are needed to determine the clinical utility of the proposed method.

Short-term clinical outcomes and predicted cost savings of dd-cfDNA-led surveillance after pediatric heart transplantation.

Endomyocardial biopsy (EMB)-led surveillance is common after pediatric heart transplantation (HT), with some centers performing periodic surveillance EMBs indefinitely after HT. Donor derived cell-free DNA (dd-cfDNA)-led surveillance offers an alternative, but knowledge about its clinical and economic outcomes, both key drivers of potential utilization, are lacking. Using single-center recipient and center-level data, we describe clinical outcomes prior to and since transition from EMB-led surveillance to dd-cfDNA-led surveillance of pediatric and young adult HT recipients. These data were then used to inform Markov models to compare costs between EMB-led and dd-cfDNA-led surveillance strategies. Over 34.5 months, dd-cfDNA-led surveillance decreased the number of EMBs by 81.8% (95% CI 76.3%-86.5%) among 120 HT recipients (median age 13.3 years). There were no differences in the incidences of graft loss or death among all recipients followed at our center prior to and following implementation of dd-cfDNA-led surveillance (graft loss: 2.9 vs. 1.5 per 100 patient-years; p = .17; mortality: 3.7 vs. 2.2 per 100 patient-years; p = .23). Over 20 years from HT, dd-cfDNA-led surveillance is projected to cost $8545 less than EMB-led surveillance. Model findings were robust in sensitivity and scenario analyses, with cost of EMB, cost of dd-cfDNA testing, and probability of elevated dd-cfDNA most influential on model findings. dd-cfDNA-led surveillance shows promise as a less invasive and cost saving alternative to EMB-led surveillance among pediatric and young adult HT recipients.

Donor-Derived Cell-Free DNA for Personalized Immunosuppression in Renal Transplantation.

The long-term outcomes of solid organ transplantation remain suboptimal. Therefore, appropriate biomarkers are needed in addition to immunosuppressive drugs and other traditional approaches for graft monitoring to achieve personalized immunosuppression and reduce premature graft loss. Donor-derived cell-free DNA (dd-cfDNA) is a minimally invasive biomarker of cell death due to graft injury. It can be quantified using droplet digital polymerase chain reaction and next-generation sequencing. Fractional dd-cfDNA determination can be affected by changes in recipient cfDNA, such as those caused by leukopenia or infection, leading to false positive or negative results, respectively. Absolute quantification of dd-cfDNA helps in overcoming this limitation. Overall, there is sufficient evidence of the clinical validity of dd-cfDNA. It detects rejection episodes early at an actionable stage and reflects the severity of graft injury without being rejection-specific. Owing to its high negative predictive value, dd-cfDNA is very useful for ruling out graft injury. Dd-cfDNA complements histological findings and can help in avoiding unnecessary biopsies. It indicates a response to rejection treatment and detects under-immunosuppression. Monitoring changes in dd-cfDNA over time may be helpful in adapting immunosuppression to prevent graft rejection. Moreover, serial dd-cfDNA determination may increase the effectiveness of transplant recipient surveillance and facilitate personalized immunosuppression when combined with other relevant clinical and diagnostic findings.

Clinical features and allograft failure rates of pulmonary antibody-mediated rejection categories.

Pulmonary antibody-mediated rejection (AMR) consensus criteria categorize AMR by diagnostic certainty. This study aims to define the clinical features and associated outcomes of these recently defined AMR categories. Adjudication committees reviewed clinical data of 335 lung transplant recipients to define clinical or subclinical AMR based on the presence of allograft dysfunction, and the primary endpoints, time from transplant to allograft failure, a composite endpoint of chronic lung allograft dysfunction and/or death. Clinical AMR was subcategorized based on diagnostic certainty as definite, probable or possible AMR if 4, 3, or 2 characteristic features were present, respectively. Allograft injury was assessed via plasma donor-derived cell-free DNA (ddcfDNA). Risk of allograft failure and allograft injury was compared for AMR categories using regression models. Over the 38.5 months follow-up, 28.7% of subjects developed clinical AMR (n=96), 18.5% developed subclinical AMR (n=62) or 58.3% were no AMR (n=177). Clinical AMR showed higher risk of allograft failure and ddcfDNA levels compared to subclinical or no AMR. Clinical AMR included definite/probable (n=21) or possible AMR (n=75). These subcategories showed similar clinical characteristics, ddcfDNA levels, and risk of allograft failure. However, definite/probable AMR showed greater measures of AMR severity, including degree of allograft dysfunction and risk of death compared to possible AMR. Clinical AMR showed greater risk of allograft failure than subclinical AMR or no AMR. Subcategorization of clinical AMR based on diagnostic certainty correlated with AMR severity and risk of death, but not with the risk of allograft failure.

Comparison of methods for donor-derived cell-free DNA quantification in plasma and urine from solid organ transplant recipients.

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. 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. The HTS method presented here showed a strong correlation of the %dd-cfDNA with ddPCR (R 2 = 0.98) and AlloSeq® cfDNA (R 2 = 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. 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.

Application of plasma donor-derived cell free DNA for lung allograft rejection diagnosis in lung transplant recipients

BackgroundDonor-derived cell-free DNA (dd-cfDNA) has been applied to monitor acute rejection (AR) in kidney and heart transplantation. This study was aimed to investigate the application of dd-cfDNA levels in the diagnosis of AR and chronic lung allograft dysfunction (CLAD) among the lung transplantation recipients (LTRs).MethodsOne hundred and seventy LTRs were enrolled at the First Affiliated Hospital of Guangzhou Medical University between 1 June 2015 and 30 March 2021. Patients were divided into 4 groups: stable group, AR group, infection group and CLAD group. The level of dd-cfDNA was analyzed using target region sequencing and the performance characteristics of dd-cfDNA for diagnosis of AR and CLAD were determined, respectively.ResultsKruskal–Wallis test showed that there were some significant differences in the level of dd-cfDNA (%) among the 4 groups, with p < 0.001. Among them, the level of dd-cfDNA (%) was highest (median 2.17, IQR [1.40–3.82]) in AR group, and higher in CLAD group (median 1.07, IQR [0.98–1.31]), but lower in infection group (median 0.71, IQR [0.57–1.07]) and lowest in stable group (median 0.71, IQR [0.61–0.84]). AUC-ROC curve analysis showed that the threshold of dd-cfDNA for AR was 1.17%, with sensitivity being 89.19% and specificity being 86.47%, and the optimal threshold of 0.89% was determined of CLAD, with sensitivity being 95.00% and specificity of 76.99%.ConclusionsPlasma dd-cfDNA could be a useful tool for the assessment of lung allograft rejection, including AR and CLAD, and holds promise as a noninvasive biomarker for “allograft injury” in both acute and chronic rejection following lung transplantation.

Role of dd-cfDNA in Detection of Subclinical Rejection in Pediatric Kidney Transplant Recipients

Background: Detection of subclinical rejections has been a real challenge for pediatric nephrology. We hypothesized that donor-derived cell-free DNA (dd-cfDNA) in combination with serum creatinine levels and a kidney biopsy provide a “gold-standard” for clinicians so that treatment for subclinical rejection can be initiated appropriately. Design/Methods: We performed a two-year cohort study on total (n=5) pediatric patients aged 5-19 years who received kidney transplant within 2015-2019. Blood was collected for dd-cfDNA, i.e., AlloSure, at the time of scheduled surveillance visits or when clinically indicated. During the study period, serum creatinine and tacrolimus were measured as well. For diagnosing subclinical rejection, dd-cfDNA was divided into 3 groups: low dd-cfDNA &lt;0.5%, high dd-cfDNA 0.5%-1%, very high dd-cfDNA&gt;1%. A kidney biopsy was performed in one patient who had very high dd-cfDNA 2.3% (nl&lt;0.2%), high serum creatinine (sCr 1.17 mg/dL, baseline 0.5 mg/dL), in the absence of tacrolimus levels. Kidney biopsy revealed acute cellular rejection (ACR) type 1A. Patient received intravenous immune globulin (IVIG) 2 g/kg x 1, IV pulses with methylprednisolone 20 mg/kg/dose x 3, followed by a steroid taper over one month. Her serum creatinine remains normal since. Whereas in the other 4 patients, dd-cfDNA did not show significant change, no subclinical rejection was observed. Conclusion: dd-cfDNA in combination with serum creatinine levels and a kidney biopsy can be considered the “gold standard” which improves early diagnostic utility for subclinical rejection.

Donor-Derived Cell-Free DNA and Active Rejection in Renal Allografts.

Renal allograft rejection contributes to significant morbidity and graft loss. In this setting, early detection of rejection is of paramount importance, which currently relies on histopathology. A reliable non-invasive marker to predict rejection would make surveillance and decision-making easier. Donor-derived cell-free DNA (dd-Cf-DNA) has recently been reported as an emerging tool to predict rejection noninvasively. The utility of cell-free DNA in clinical practice has so far not been studied in an Indian setting. As it offers direct clinical application, we have chosen to investigate this biomarker as a tool to predict rejection. A pilot study with convenient sample size was designed, as this is the first of its kind study so far reported from India. Patients being evaluated with a graft biopsy for graft dysfunction were included. Patients with stable graft function, defined as creatinine within 10% of their best creatinine and no proteinuria for the preceding 12 months, were also included. Ten milliliters of whole blood from each of the recipients was collected in DNA isolation tubes. Two milliliters of donor blood was also obtained in ethylenediaminetetraacetic acid (EDTA) tubes. All recipients also provided a buccal swab. Total cell-free DNA was extracted from 2 ml of recipient plasma using circulating DNA isolation kit. Upon identification of the donor-specific DNA marker for each of the patients from the paired donor sample, presence of the cell-free DNA fraction in the recipient's plasma was detected and quantified. Renal biopsy reports and clinical details were also recorded. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were analyzed. Receiver operating characteristic (ROC) curve analysis was also performed. A total of 31 patients were recruited. Twenty patients underwent graft biopsies for graft dysfunction, of which 12 patients had features of active rejection and eight had nonrejection causes of graft dysfunction. Eleven patients with stable graft were included in the study. In our study, dd-Cf-DNA performed best in predicting antibody-mediated rejection (ABMR) and higher grades of T-cell-mediated rejection (TCMR) (1B). It did not detect TCMR 1A accurately. It serves as a good marker to rule out rejection. It gave a NPV of 100% for TCMR 1B or ABMR, 100% for ABMR alone, and 81% for any rejection. dd-Cf-DNA percentages outperform absolute concentrations in their discriminatory ability. We have demonstrated the diagnostic accuracy of dd-Cf-DNA in predicting active rejection of the renal allograft. It performs well in ABMR and higher grades of TCMR. This is the first of its kind study reported from India, to the best of our knowledge. This tool serves as a good rule out test for ABMR and higher grades of TCMR. It performs poorly in TCMR 1A.

Donor-derived cell free DNA (dd-cfDNA) in pregnant kidney transplant recipients

Distinguishing between allograft rejection, kidney disease progression, and preeclampsia in a non-invasive manner can be difficult in pregnant kidney transplant recipients. Donor-derived cell-free DNA (dd-cfDNA) is a biomarker that can be measured non-invasively to assess risk of allograft rejection. However, using dd-cfDNA to monitor allograft status in pregnant kidney transplant recipients requires distinguishing between fetal, donor, and recipient cfDNA fragments. In this study, we aimed to measure dd-cfDNA in a cohort of pregnant kidney transplant recipients using a combination of NIPT and transplant monitoring technology utilizing cfDNA.

Understanding Donor-derived Cell-free DNA in Kidney Transplantation: An Overview and Case-based Guide for Clinicians.

Kidney transplant recipients undergo lifelong monitoring of allograft function and evaluation for transplant complications. The current monitoring paradigm utilizes blood, urine, and tissue markers that are insensitive, nonspecific, or invasive to obtain. As a result, problems are detected late, after significant damage has accrued, and often beyond the time at which complete resolution is possible. Indeed, most kidney transplants eventually fail, usually because of chronic rejection and other undetected injury. There is a clear need for a transplant-specific biomarker that enables a proactive approach to monitoring via early detection of reversible pathology. A biomarker that supports timely and personalized treatment would assist in achieving the ultimate goal of improving allograft survival and limiting therapeutic toxicity to the recipient. Donor-derived cell-free DNA (ddcfDNA) has been proposed as one such transplant biomarker. Although the test is presently utilized most in the United States, it is conceivable that its use will become more widespread. This review covers aspects of ddcfDNA that support informed use of the test by general nephrologists, including the basic biology of ddcfDNA, methodological nuances of testing, and general recommendations for use in the kidney transplant population. Clinical contexts are used to illustrate evidence-supported interpretation of ddcfDNA results and subsequent management. Finally, knowledge gaps and areas for further study are discussed.

Increase of donor-derived cell-free DNA (dd-cfDNA) in coronary allograft vasculopathy in heart transplanted patients

Increase of donor-derived cell-free DNA (dd-cfDNA) in coronary allograft vasculopathy in heart transplanted patients