Adapting an Established Clinical Chemistry Quality Control Measure for Droplet Generation Performance in Digital PCR

To the Editor: Droplet digital PCR (ddPCR)1 is an emerging platform that is being increasingly adopted by clinical laboratories. The partitioning of a PCR reaction into discrete droplets enables the accurate detection and quantification of molecular targets. Single molecule analysis by this manner is accurate, cost-effective, and readily performed compared with next-generation sequencing and mass spectrometry. The typical ddPCR workflow comprises 4 key steps: ( a ) preparation of the PCR reaction, ( b ) droplet generation (DG), ( c ) endpoint PCR, and ( d ), droplet analysis. The process of DG is a critical step of the workflow because this step is considered to be most prone to droplet loss (1). It is thus important to recognize that a loss of droplets during DG will affect the number of droplets recoverable for droplet analysis in an optimized assay. Consequently, lower droplet numbers can lead to a loss of analytical sensitivity and may confound the evaluation of samples for molecular targets that are present in very low concentrations. To date, informative measures to evaluate DG performance within the ddPCR workflow remain unaddressed. The Levey–Jennings chart in conjunction with the “Westgard rules” is the staple of quality management in clinical chemistry measurement systems. Here, we outline an approach that uses the Levey–Jennings chart and a set of adapted Westgard rules to monitor DG performance in …