Absolute or Relative Quantification of Donor-derived Cell-free DNA in Kidney Transplant Recipients: Case Series

Abstract

Background. Donor-derived cell-free DNA (dd-cfDNA) is increasingly recognized as a valuable biomarker for acute transplant injury, with possible indications in the detection of cellular or humoral rejection and the guidance of immunosuppressive therapy. There is an ongoing debate on whether relative or absolute quantification of dd-cfDNA is more reliable for the detection of acute transplant injury. Methods. We retrospectively reviewed all 22 kidney transplant recipients who underwent dd-cfDNA measurements (percentage and absolute) between April 2020 and April 2021 at our institution. Of these, 9 (41%) showed discrepancies between absolute (cutoff: 50 copies/mL) and relative (cutoff: 0.5%) quantification in at least 1 dd-cfDNA measurement. Results. We report on 9 of 22 cases with discrepancies in relative and absolute quantification of dd-cfDNA, which were predominantly late posttransplant patients. We found bacterial and viral infections, as well as low leukocyte count from chronic myeloid leukaemia treatment, to be reasons for variability in total cell-free DNA (cfDNA), leading to inter- and intraindividual variability in relative dd-cfDNA quantification. When correlating dd-cfDNA quantification and biopsy results, as well as clinical course, our data indicate that relying solely on relative dd-cfDNA can lead to false-negative and false-positive results. Conclusions. In summary, these cases argue that absolute quantification of dd-cfDNA is better suited in patients with underlying conditions affecting total cfDNA levels and suggest using both absolute and relative dd-cfDNA together for higher reliability and interindividual comparability in the clinical setting. Especially for patients with chronic active antibody–mediated rejection, further studies on the use of dd-cfDNA are desirable.

Antibody-mediated Rejection Without Detectable Donor-specific Antibody Releases Donor-derived Cell-free DNA: Results From the Trifecta Study

“…Most analyses in this study focused on the %dd-cfDNA, either as continuous numbers or using cutoffs (the historical diagnostic cutoff of ≥1%). However, because recent studies have suggested that actual dd-cfDNA quantity may also be diagnostically useful, 15 , 32 in logistic regression, we assessed the predictive ability of the quantitative dd-cfDNA measurement (copies per mL), as well as %dd-cfDNA. All samples were analyzed, irrespective of time posttransplant and other potential confounders (eg, cancer), and thus the predictions are not intended to simulate the clinical results when a variety of exclusions are applied.…”

Donor-derived cell-free DNA as a diagnostic tool in transplantation

“…A problem with fractional dd-cfDNA determination is that it is influenced by variations in total cf-DNA levels due to leukopenia, leukocytosis and inflammatory illness; leading to falsely elevated or decreased results (Whitlam et al, 2019;Osmanodja et al, 2021). Of particular importance is the systematic increase of dd-cfDNA percentages when CNI are tapered, due to an influence of CNI on leucocyte stability (Schuetz et al, 2020).…”

“…Of particular importance is the systematic increase of dd-cfDNA percentages when CNI are tapered, due to an influence of CNI on leucocyte stability (Schuetz et al, 2020). In clinical practice a combination of absolute quantification and fractional abundance of dd-cfDNA with consideration of total cfDNA as well seems to provide the most comprehensive diagnostic information (Oellerich et al, 2019;Whitlam et al, 2019;Oellerich et al, 2021;Osmanodja et al, 2021).…”

Donor-Derived Cell-free DNA for Personalized Immunosuppression in Renal Transplantation

“…10,28 The major pitfalls of fractional determination methods include false-positive or false-negative results due to any rejection unrelated decrease or increase in recipient DNA. 10,16,30,31 Absolute quantification by ddPCR is not affected by these changes. 12 Variations in the efficiency of cfDNA extraction and ddPCR amplification should be considered.…”