TRANSPLANT MONITORING
Accurate and timely detection of allograft rejection, along with effective immunosuppression, is critical for ensuring long-term graft survival
cfDNA pilot workflow methodology Despite widespread acceptance of cfDNA as a biomarker for organ rejection in the US and Europe, the UK has yet to include dd-cfDNA testing in clinical guidelines.11
Aware of the burden that
allograft rejection has on the health service, the team at NBT is keen to help change that. If the cfDNA pilot study is successful it is hoped that this methodology will be included in national technology adoption frameworks. The workflow in this pilot study includes the use of Promega’s new Maxwell RSC Rapid circulating cfDNA extraction kits. Key to the success of the study would be the quality, yield and consistency of DNA, which the team has been achieving with the Maxwell DNA extraction kits they routinely use for HLA typing. The Maxwell instrument, as stated by Sarinder Day, was adopted for its reliability and consistent high-quality DNA yield.
The workflow methodology of the cfDNA trial centres on the downstream use of digital droplet PCR. In essence plasma would be collected from transplant patients immediately after transplant and at another interval a few months after implantation to allow the initial levels of dd-cfDNA to settle. In kidney transplantation, dd-cfDNA in the blood is usually highest immediately after
Analysis of cfDNA from the bloodstream has been widely embraced as a minimally invasive method of assessing a patient’s physiological state.
transplantation and generally decreases. It has been reported that percentage of dd-cfDNA generally decreases to approximately 0.5% within 10 days.6,13,14 The ideal frequency of testing in
different transplant settings would need to be investigated to define optimal diagnostic thresholds, with factors such as organ type transplanted, the time from transplantation, coexisting conditions, and overall clinical context being considered. Plasma is collected in Streck tubes and cfDNA would then be extracted using the Promega Maxwell RSC Rapid ccfDNA extraction kits. The resulting cfDNA is analysed using digital droplet PCR and around 45 SNP targets separating out distinctive alleles to ensure suitable discrimination between donor and patient. The output provides both absolute quantification and fractional abundance of dd-cfDNA.
The team is currently working with the clinical team to identify control groups and patients who they suspect might be going through rejection post-transplant. Similar pilot studies are being carried out at other H&I test sites looking at other technologies including NGS.
Future directions and clinical implementation
If this pilot study is successful, it could pave the way for the much- needed improvement of personalised immunosuppression necessary in kidney transplantation to reduce premature graft loss.2
It would add crucial evidence to the significant body of work demonstrating that levels of circulating dd-cfDNA are strongly associated with the presence, activity and severity of kidney allograft rejection and that assessment of circulating dd-cfDNA improves the
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