Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal.

The field of liquid biopsy has seen extensive growth in recent decades, making it one of the most promising areas in molecular diagnostics. Circulating cell-free DNA (ccfDNA) especially is used as an analyte in a growing number of diagnostic assays. These assays require specified preanalytical workflows delivering ccfDNA in qualities and quantities that facilitate correct and reliable results. As each step and component used in the preanalytical process has the potential to influence the assay sensitivity and other performance characteristics, it is key to find an unbiased experimental setup to test these factors in diagnostic or research laboratories. We defined one such setup by using blood from healthy subjects and commercially available products for blood collection, spike-in material, ccfDNA isolation, and qPCR assays. As the primary read-out, we calculated the probit model-based LOD95 (limit of detection of the 95th percentile) from the qPCR assay results. In a proof of principle study we tested two different but widely used blood ccfDNA profile stabilization technologies in blood collection tubes, the Cell-Free DNA BCT and the PAXgene Blood ccfDNA Tube. We tested assays for three different EGFR gene mutations and one BRAF gene mutation. The study design revealed differences in performance between the two tested technologies for all four mutations. In conclusion, we successfully established a blueprint for a test procedure capable of verifying and validating a liquid biopsy workflow from blood collection to the analytical result.