BACKGROUND: Cell-free DNA (cfDNA) from grafts in the circulation of transplant recipients is a potential biomarker of rejection. Its usefulness was investigated after heart transplantation during the maintenance phase by use of microarrays and massive parallel sequencing of donor and recipient DNA. Disadvantages of these methods are high costs, long turnaround times, and need for donor DNA. Therefore, we sought to develop a rapid and cost-effective method using digital droplet PCR (ddPCR).

This is an open access article under the terms of the Creat ive Commo ns Attri bution-NonCo mmercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive biomarker for comprehensive monitoring of allograft injury and rejection in kidney transplantation (KTx). dd-cfDNA quantification of copies/mL plasma (dd-cfDNA[cp/mL]) was compared to dd-cfDNA fraction (dd-cfDNA[%]) at prespecified visits in 189 patients over 1 year post KTx. In patients (N = 15, n = 22 samples) with biopsy-proven rejection (BPR), median dd-cfDNA(cp/mL) was 3.3-fold and median dd-cfDNA(%) 2.0-fold higher (82 cp/ mL; 0.57%, respectively) than medians in Stable Phase patients (N = 83, n = 408) without rejection (25 cp/mL; 0.29%). Results for acute tubular necrosis (ATN) were not significantly different from those with biopsy-proven rejection (BPR). dd-cfDNA identified unnecessary biopsies triggered by a rise in plasma creatinine. Receiver operating characteristic (ROC) analysis showed superior performance (P = .02) of measuring dd-cfDNA(cp/mL) (AUC = 0.83) compared to dd-cfDNA(%) (area under the curve [AUC] = 0.73). Diagnostic odds ratios were 7.31 for dd-cfDNA(cp/mL), and 6.02 for dd-cfDNA(%) at thresholds of 52 cp/mL and 0.43%, respectively. Plasma creatinine showed a low correlation (r = 0.37) with dd-cfDNA(cp/mL). In a patient subset (N = 24) there was a significantly higher rate of patients with elevated dd-cfDNA(cp/ mL) with lower tacrolimus levels (<8 μg/L) compared to the group with higher tacrolimus concentrations (P = .0036) suggesting that dd-cfDNA may detect inadequate immunosuppression resulting in subclinical graft damage. Absolute dd-cfDNA(cp/mL) allowed for better discrimination than dd-cfDNA(%) of KTx patients with BPR and is useful to avoid unnecessary biopsies.

BackgroundGraft-derived cell-free DNA (GcfDNA), which is released into the blood stream by necrotic and apoptotic cells, is a promising noninvasive organ integrity biomarker. In liver transplantation (LTx), neither conventional liver function tests (LTFs) nor immunosuppressive drug monitoring are very effective for rejection monitoring. We therefore hypothesized that the quantitative measurement of donor-derived cell-free DNA (cfDNA) would have independent value for the assessment of graft integrity, including damage from acute rejection.Methods and findingsTraditional LFTs were performed and plasma GcfDNA was monitored in 115 adults post-LTx at three German transplant centers as part of a prospective, observational, multicenter cohort trial. GcfDNA percentage (graft cfDNA/total cfDNA) was measured using droplet digital PCR (ddPCR), based on a limited number of predefined single nucleotide polymorphisms, enabling same-day turn-around. The same method was used to quantify blood microchimerism. GcfDNA was increased >50% on day 1 post-LTx, presumably from ischemia/reperfusion damage, but rapidly declined in patients without graft injury within 7 to 10 d to a median <10%, where it remained for the 1-y observation period. Of 115 patients, 107 provided samples that met preestablished criteria. In 31 samples taken from 17 patients during biopsy-proven acute rejection episodes, the percentage of GcfDNA was elevated substantially (median 29.6%, 95% CI 23.6%–41.0%) compared with that in 282 samples from 88 patients during stable periods (median 3.3%, 95% CI 2.9%–3.7%; p < 0.001). Only slightly higher values (median 5.9%, 95% CI 4.4%–10.3%) were found in 68 samples from 17 hepatitis C virus (HCV)–positive, rejection-free patients. LFTs had low overall correlations (r = 0.28–0.62) with GcfDNA and showed greater overlap between patient subgroups, especially between acute rejection and HCV+ patients. Multivariable logistic regression modeling demonstrated that GcfDNA provided additional LFT-independent information on graft integrity. Diagnostic sensitivity and specificity were 90.3% (95% CI 74.2%–98.0%) and 92.9% (95% CI 89.3%–95.6%), respectively, for GcfDNA at a threshold value of 10%. The area under the receiver operator characteristic curve was higher for GcfDNA (97.1%, 95% CI 93.4%–100%) than for same-day conventional LFTs (AST: 95.7%; ALT: 95.2%; γ-GT: 94.5%; bilirubin: 82.6%). An evaluation of microchimerism revealed that the maximum donor DNA in circulating white blood cells was only 0.068%. GcfDNA percentage can be influenced by major changes in host cfDNA (e.g., due to leukopenia or leukocytosis). One limitation of our study is that exact time-matched GcfDNA and LFT samples were not available for all patient visits.ConclusionsIn this study, determination of GcfDNA in plasma by ddPCR allowed for earlier and more sensitive discrimination of acute rejection in LTx patients as compared with conventional LFTs. Potential blood microchimerism was quantitatively low and had no significant influence on GcfDNA value. Further r...

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A double-blind, parallel-group, triple-dummy-designed, single-oral-dose study compared the efficacy, tolerability, safety, and dose-response of 5 mg/kg (n = 32) and 10 mg/kg (n = 28) ibuprofen suspension, 10 mg/kg acetaminophen elixir (n = 33), and placebo liquids (n = 34) in 127 children (2 to 11 years of age) with fever (101 degrees to 104 degrees F). Blood samples, oral temperatures, pulse, blood pressure, and respiration were obtained before and 1/2, 1, 2, 3, 4, 5, 6, and 8 hours after the dose was administered. The study was terminated early if oral temperature was greater than 104 degrees F or if it increased 1 degree F above baseline. All agents were well tolerated and more effective than placebo (p less than 0.05) for fever control. Ibuprofen, 10 mg/kg, was favored over 10 mg/kg acetaminophen (p less than 0.05). For temperatures greater than 102.5 degrees F, a dose-response relationship for 5 and 10 mg/kg ibuprofen was demonstrated in terms of percentage of fever reduction and in terms of the initial 2-hour rate of decrease in temperature. Antipyretic efficacy for temperatures greater than 102.5 degrees F was 10 mg/kg ibuprofen greater than 5 mg/kg greater than 10 mg/kg acetaminophen greater than placebo. All treatments were well tolerated. No significant clinical or laboratory abnormalities were noted. Ibuprofen suspension may be a safe and effective antipyretic in children.