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Background Non-invasive molecular analysis of cell-free DNA (cfDNA) became a sensitive biomarker for monitoring organ transplantation or for detection of fetal DNA (cffDNA) in noninvasive prenatal test. In this study, we compared the efficiencies of four (semi)-automated cfDNA isolation instruments using their respective isolation kit: MagNA Pure 24 (Roche®), IDEAL (IDSolution®), LABTurbo 24 (Taigen®) and Chemagic 360 (Perkin Elmer®). The cfDNA was isolated from 5 plasma samples and the Rhesus D (RhD)-cffDNA from 5 maternal plasmas. The cfDNA were quantified by digital droplet PCR (ddPCR), BIABooster system and QUBIT fluorometer. The cfDNA fragment size profiles were assessed by BIABooster system. Chimerism were quantified by home-made ddPCR and Devyser NGS kit. RhD-cffDNA in maternal plasma were detected between weeks 14 and 24 of amenorrhea using free DNA Fetal RHD Kit® (Biorad®). Results Statistical tests have shown differences in DNA yield depending on the isolation procedure and quantification method used. Magna Pure isolates smaller cfDNA fragment size than other extraction methods (90% ± 9% vs. 74% ± 8%; p = 0.009). Chimerism was only reliable from LABTurbo 24 extractions using the NGS but not with ddPCR whatever extraction methods. RhD-cffDNA were detected by all isolation methods, although IDEAL and LABTurbo 24 systems seemed more efficient. Conclusions This comparative study showed a dependency of cfDNA yield depending on isolation procedure and quantification method used. In total, these results suggest that the choice of pre-analytical isolation systems needs to be carefully validated in routine clinical practice.

BackgroundQuantification of chimerism showing the proportion of the donor in a recipient is essential for the follow-up of hematopoietic stem cell transplantation but can also be useful to document an immune tolerance situation after solid organ transplantation. Historically, chimerism has been quantified from genomic DNA, but with technological advances, chimerism from donor-derived cell-free DNA seems particularly relevant in solid organ transplantation.MethodsThe reference method was until recently the short tandem repeat technique, but new innovative techniques as digital PCR (dPCR) and NGS, have revolutionized the quantification of chimerism, such as the so-called microchimerism analysis. After a short review of chimerism methods, a comparison of chimerism quantification data for two new digital PCR systems (QIAcuity™ dPCR (Qiagen®) and QuantStudio Absolute Q (ThermoFisher®) and two NGS-based chimerism quantification methods (AlloSeq HCT™ (CareDx®) and NGStrack™ (GenDX®)) was performed.ResultsThese new methods were correlated and concordant to routinely methods (r²=0.9978 and r²=0.9974 for dPCR methods, r²=0.9978 and r²=0.9988 for NGS methods), and had similar high performance (sensitivity, reproductibility, linearity).ConclusionFinally, the choice of the innovative method of chimerism within the laboratory does not depend on the analytical performances because they are similar but mainly on the amount of activity and the access to instruments and computer services.

BackgroundMany studies have reported the relevance of donor-derived cfDNA (dd-cfDNA) after lung transplantation (LTx) to diagnose and monitor acute rejection (AR) or chronic rejection or infection (INF). However, the analysis of cfDNA fragment size has not been studied. The aim of this study was to determine the clinical relevance of dd-cfDNA and cfDNA size profiles in events (AR and INF) during the first month after LTx.MethodsThis prospective, single-center study includes 62 LTx recipients at the Marseille Nord Hospital, France. Total cfDNA quantification was performed by fluorimetry and digital PCR, dd-cfDNA by NGS (AlloSeq cfDNA-CareDX®), and the size profile by BIABooster (Adelis®). A bronchoalveolar lavage and transbronchial biopsies at D30 established the following groups: not-injured and injured graft (AR, INF, or AR+INF).ResultsQuantification of total cfDNA was not correlated with the patient’s status at D30. The percentage of dd-cfDNA was significantly higher for injured graft patients at D30 (p=0.0004). A threshold of 1.72% of dd-cfDNA correctly classified the not-injured graft patients (negative predictive value of 91.4%). Among recipients with dd-cfDNA >1.72%, the quantification of small sizes (80-120bp) >3.70% identified the INF with high performance (specificity and positive predictive value of 100%).ConclusionWith the aim of considering cfDNA as a polyvalent non-invasive biomarker in transplantation, an algorithm combining the quantification of dd-cfDNA and small sizes of DNA may significantly classify the different types of allograft injuries.

Introduction: Donor-derived cell-free DNA (dd-cfDNA) emerged as a candidate biomarker for detecting graft injury, particularly from antibody-mediated rejection, and is currently proposed to complement donor-specific antibodies (DSAs) as an alloimmune-mediated injury surveillance method. We sought to investigate the impact of an initial increase of dd-cfDNA level on subsequent decline in graft function. Materials:The study included all kidney transplant (KT) recipients that underwent dd-cfDNA testing as part of their clinical care between September 2017 and December 2019 at our center. Only patients with a follow-up of at least 12 months were included in this analysis. An elevated dd-cfDNA was defined as a level over 0.5%. Results: Of the 171 KT recipients tested for dd-cfDNA, 49 were followed for at least 12 months since initial testing. The study cohort had an initial serum creatinine and eGFR of 1.64 ± 0.58 mg/dl and 48 ± 21 ml/min/1.73m2, respectively. Overall, the absolute and percentual decline of eGFR were -0.12 ml/min/month (IQR, -0.43 to 0.56) and -4.26% (IQR, -20.5% to 17%), with 26.5% and 10.2% of patients having an eGFR decline of more than 15% and 30%, respectively. Fifteen patients (30.6%) had an elevated dd-cfDNA (over 0.5%) and had a similar baseline allograft function compared to those without an elevated dd-cfDNA level. After a median follow-up period of 15.3 months (IQR:13.6-19), patients with elevated dd-cfDNA had a faster decline of eGFR [absolute eGFR decline, -0.21 ml/min/month (IQR, -0.36 to 0.22); percentual GFR decline, -8.7% (IQR, -25% to 4.4%)], compared to those without elevated dd-cfDNA [absolute eGFR decline, +0.03 ml/min/month (IQR, -0.5 to 0.6); percentual GFR decline, +1.14% (IQR, -14% to 28%)] (p=0.09)(Figure 1). Similarly, there was a tendency for a higher percentage of patients with elevated dd-cfDNA for a decline of eGFR greater than 15% (33.3% vs. 23.5%, p=0.5) or 30% (13.3% vs. 8.8%, p=0.6), respectively. In addition, patients with DSAs and an elevated dd-cfDNA showed a greater percentual eGFR decline (-9.1±16.5%), compared to those with DSAs and normal dd-cfDNA (+3.3±21.1%), or to those without DSAs and normal dd-cfDNA (+2.3±18.05%). In multivariable logistic regression analysis, an elevated dd-cfDNA level was independently associated with the subsequent percentual decline of eGFR (OR, 0.96; 95%CI, 0.93 to 1.00; p=0.05). Conclusion:We have shown that despite initial stable allograft function, a higher dd-cfDNA level associates with subsequent decline of eGFR. Thus, the initial identification of subclinical graft injury could associate with longterm graft outcomes, expanding the clinical utility of dd-cfDNA.