Digital PCR (dPCR) has gained popularity in recent years for cancer research such as rare mutation detection, minimal residual disease, treatment selection, and recurrence monitoring. However, currently available technologies suffer from several limitations such as tedious workflow, long time-to-result, poor multiplexity, and inconsistent reagent digitization that severely hindered its broad adoption. We have designed and manufactured a dPCR platform that is capable of consistent sample digitization, thermal cycling and simultaneous interrogation of 20,000 partitions with walk-away workflow. The instrument integrates a loading and digitization manifold, a thermal cycler and an imaging engine with 5 optical channels into a single benchtop box. The Microfluidic Array Partitioning (MAP) consumable is a micro-injection molded, automation-ready plate that contains 16 individual units that can be loaded simultaneously. Each unit partitions an individual sample into 20,000 fluidically isolated partitions while utilizing >90% of inputted sample. We utilized an oncology standard assay, confocal microscopy, and real-time imaging to validate the dynamic range, sample utilization, and digitization consistency of the platform, respectively. Using the European Breast Cancer Standards ERM-AD623 we quantified replicates that were serially diluted from 1 × 105cp/μL down to 10cp/μL. We demonstrated that the dPCR platform can consistently quantify targets over 5-orders-of-magnitude of concentration with high consistency in both quantification and partition numbers. With confocal microscopy, the reagent inside the partitions are visualized to characterize sample utilization. Using conventional qPCR calibration dye ROX and real-time imaging, we show that over 99% of the partitions can be consistently analyzed for high quantification confidence. With the combination of ease-of-use, fast time-to-result and consistent performance, we believe this novel dPCR platform provides unparalleled usability and capability and will allow dPCR to fulfill its promise to impact cancer patient care.

Citation Format: Robert Lin, Andrew Zayac, Steve Gallagher, Lingxia Jiang, Felicia Linn, Paul Hung. Fully integrated single instrument imaging-based digital PCR platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 43.