Clinical relevance of cell-free DNA quantification and qualification during the first month after lung transplantation.

Background: Many 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. Methods: This 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). Results: Quantification 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%). Conclusion: With 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.

New methods for the quantification of mixed chimerism in transplantation.

Background: Quantification 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. Methods: The 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. Results: These 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). Conclusion: Finally, 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.

Qualitative and quantitative comparison of cell-free DNA and cell-free fetal DNA isolation by four (semi-)automated extraction methods: impact in two clinical applications: chimerism quantification and noninvasive prenatal diagnosis.

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.

Evaluation of Next-Generation Sequencing and Crystal Digital PCR for Chimerism Monitoring of Post-Allogeneic Hematopoietic Stem Cell Transplantation.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for most hematologic diseases. To evaluate the level of donor engraftment, chimerism must be carefully monitored after HSCT. Short tandem repeats, quantitative PCR (qPCR), and, more recently, digital PCR (dPCR) are widely used to determine the proportions of donor and recipient cells after HSCT. The screening and quantification of chimerism have been evaluated by 2 new methods: a ready-to-use next-generation sequencing (NGS)-based method using the Devyser ChimerismNGS kit and an original combination of the Stilla crystal digital PCR (cdPCR) platform with 3-color multiplexing capacity using GenDX KMRtrack reagents. The genotyping of 4 HSCT pairs by cdPCR using 11 triplex mixes of the GenDX KMRtype kit was consistent at 98.8% with qPCR. Informative samples (n = 20) from 6 donor-recipient pairs and 1 external proficiency test demonstrated the reliability of the results (0.1% to 50%) for the 2 methods. The methods are also highly sensitive (0.1%) and accurate. The chimerism values of the 2 methods are correlated and concordant with those of the reference methods. In addition, the ADVYSER software (Devyser) is user-friendly and well adapted to chimerism monitoring. In conclusion, these 2 innovative methods are easy to perform and user-friendly in all molecular, hematology, and immunogenetic laboratories and allow the genotyping and monitoring of chimerism with high performance and sensitivity.