Introduction: Graft damage and failure are major problems after solid-organ transplantation. Biomarkers for monitoring transplanted patients provide new opportunities to substantially improve patient management, graft lifespan, and patient survival. There is strong evidence that donor-derived cell-free DNA (DD-cfDNA) increases when transplanted organs are damaged. The majority of this information is derived from tests using next-generation sequencing. Here we present an evaluation of new technology, multiplex digital PCR (mdPCR), which has the potential to revolutionize DD-cfDNA testing.

Method: This mdPCR method takes advantage of Luminex's DigiMAP multiplex PCR chemistry and the QuantStudio Absolute Q Digital PCR platform (Thermo Fisher Scientific, previously Combinati). The bench work for mdPCR is completed in less than five hours. Data is analyzed using a custom R package. Assay performance characteristics were determined using artificial mixtures of genomic DNA, and inter-laboratory reproducibility was assessed. Blood was collected from kidney and heart transplant recipients, cf-DNA was isolated using a variety of methods, and mdPCR was used to quantify DD-cfDNA levels.

Results: This methodology distinguished donor and recipient DNA and provided absolute quantification of the number of DNA copies. The following performance characteristics were determined: sensitivity, the limit of detection, and linear range, inter- and intra- assay consistency. Several factors affected performance including: DNA characteristics, probe design, primer design, and PCR reaction components. Results showed that high levels of DD-cfDNA are consistent with clinical observations and that rapid testing is feasible (< five hours).

Conclusion: The mdPCR approach has high promise to revolutionize diagnostic testing in cancer, infectious disease, and transplant monitoring. Our study demonstrates the advantages of this technology for the detection of DD-cfDNA which includes absolute quantitation, sensitivity, ease of use, short turn-around-time, and a relatively low cost when compared to next-generation sequencing techniques. A test with these features could transform the management of transplant recipients and lead to prolonged graft and patient survival.