Biological Variation of Donor-Derived Cell-Free DNA in Renal Transplant Recipients: Clinical Implications

Abstract

Background Previous studies have demonstrated that donor-derived cell-free DNA (dd-cfDNA) found in circulating blood of transplant recipients may serve as a noninvasive biomarker of allograft rejection. To better interpret the clinical meaning of dd-cfDNA, it is essential to understand the biological variation of this biomarker in stable healthy recipients. This report establishes the biological variation and clinical reference intervals of dd-cfDNA in renal transplant recipients by using an analytically validated assay that has a CV of 6.8%. Methods We sampled venous blood at patient surveillance visits (typically at posttransplant months 1–4, 6, 9, and 12) in a 14-center observational study. Patients with stable renal allograft function spanning ≥3 serial visits were selected. We used AlloSure®, a targeted next-generation sequencing-based approach, to measure dd-cfDNA in the plasma and computed the intraindividual CV (CVI) and interindividual CV (CVG), the index of individuality (II), and reference change value (RCV). Results Of 93 patients, 61% were men, 56% were Caucasian, mean age was 49 years, and 63% were deceased donor kidney recipients. Of 380 blood samples, the dd-cfDNA median value was 0.21% (interquartile range 0.12%–0.39%) and the 97.5th percentile was 1.20%. In 18 patients with an average of 4.1 tests, the CVI was 21%, CVG was 37%, II was 0.57, and RCV was 61%. Conclusions In a renal transplant recipient, a dd-cfDNA level above 1.2% is out of range and potentially abnormal. A serial increase of up to 61% in level of dd-cfDNA in a patient may be attributable to biological variation. Clinicaltrials.gov Identifier: NCT02424227

High levels of dd-cfDNA identify patients with TCMR 1A and borderline allograft rejection at elevated risk of graft injury

“…The skewness of the violin plot may represent the differentiation seen in the variation of injury, with demarcations seen at 1% and 0.74% being previously published as thresholds for defining rejection. The median dd‐cfDNA level prior to biopsy was 0.19% supporting the baseline previous published using a reference population …”

Absolute quantification of donor-derived cell-free DNA as a marker of rejection and graft injury in kidney transplantation: Results from a prospective observational study

“…So far, studies directly comparing different dd‐cfDNA determination methods are lacking. It is encouraging, however, that the median values of clinically stable patients were very similar in different studies using different methods for dd‐cfDNA determination . In our cohort, the median was 0.29% (IQR: 0.17%‐0.56%).…”

“…Bloom and Sigdel proposed a dd‐cfDNA(%) cut‐off value at 1%. The 97.5th percentile of Stable Phase patients reported by Bromberg et al was 1.20% and in our study 1.03% excluding ties in a sensitivity analysis. We suggest a cut‐off at 0.5% based on simultaneous maximization of sensitivity and specificity from our study data.…”

“…In our cohort, the median was 0.29% (IQR: 0.17%‐0.56%). Bromberg published a value of 0.21% (IQR: 0.12%‐0.39%), Bloom of 0.30% (IQR: 0.14%‐0.77%), Sigdel of 0.40%, and Gielis a mean value of 0.46% (±0.21%). Bloom and Sigdel proposed a dd‐cfDNA(%) cut‐off value at 1%.…”

Day-to-day and within-day biological variation of cell-free DNA

“…34 However, our estimations of the CV I and the RCV are comparable to values estimated for donor-derived cfDNA levels in stable renal transplant recipients measured monthly for up to one year. 35 Lastly, the observed decline in cfDNA levels during the day could be linked to higher physical activity of the participants during the day compared with the evening. However, all participants performed only sedentary tasks during the day, and thus activity was the same throughout the day and evening.…”