Evaluation of cell-free DNA approaches for multi-cancer early detection.

In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test.

High-intensity sequencing reveals the sources of plasma circulating cell-free DNA variants.

Accurate identification of tumor-derived somatic variants in plasma circulating cell-free DNA (cfDNA) requires understanding of the various biological compartments contributing to the cfDNA pool. We sought to define the technical feasibility of a high-intensity sequencing assay of cfDNA and matched white blood cell DNA covering a large genomic region (508 genes; 2 megabases; >60,000× raw depth) in a prospective study of 124 patients with metastatic cancer, with contemporaneous matched tumor tissue biopsies, and 47 controls without cancer. The assay displayed high sensitivity and specificity, allowing for de novo detection of tumor-derived mutations and inference of tumor mutational burden, microsatellite instability, mutational signatures and sources of somatic mutations identified in cfDNA. The vast majority of cfDNA mutations (81.6% in controls and 53.2% in patients with cancer) had features consistent with clonal hematopoiesis. This cfDNA sequencing approach revealed that clonal hematopoiesis constitutes a pervasive biological phenomenon, emphasizing the importance of matched cfDNA-white blood cell sequencing for accurate variant interpretation.

Microfluidic integration of Western blotting is enabled by electrotransfer-assisted sodium dodecyl sulfate dilution.

We integrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent antibody probing in a single, monolithic microdevice to realize microfluidic Western blotting. A hurdle to successful on-chip Western blotting lies in restoring antibody recognition of previously sized (denatured, reduced) proteins. To surmount this hurdle, we locally dilute free SDS from SDS-protein complexes using differential electromigration of the species during electrotransfer between SDS-PAGE and blotting regions of a microchamber. Local dilution of SDS minimizes re-association of SDS with proteins offering means to restore antibody binding affinity to proteins after SDS-PAGE. To achieve automated, programmable operation in a single instrument, we utilize a 1 × 2 mm(2) glass microchamber photopatterned with spatially distinct, contiguous polyacrylamide regions for SDS-PAGE, electrotransfer, and antibody blotting. Optimization of both the SDS-PAGE and electrotransfer conditions yields transfer distances of <1 mm (40 s). The Western blot is completed in 180 s, with fully automated assay operation using programmable voltage control. After SDS-PAGE and electrotransfer, we observe ~80% capture of protein band mass on the blotting region for a model protein, C-reactive protein. This novel microfluidic Western blot approach introduces fine transport control for in-transit protein handling to form the basis for an automated, rapid alternative to conventional slab-gel Western blotting.

Ultrashort separation length homogeneous electrophoretic immunoassays using on-chip discontinuous polyacrylamide gels.

To realize efficient homogeneous electrophoretic immunoassays, we introduce discontinuous polyacrylamide gels that enable quantitative assay completion in separation lengths as short as 350 mum in <10 s. The discontinuous cross-linked gels reduce the required electrophoretic separation lengths and thereby significantly reduce the required applied electrical potentials needed to achieve 100's V/cm electric field strengths for rapid electrophoresis. To optimize the discontinuous polyacrylamide gel assay format, we demonstrate development of a two-color homogeneous electrophoretic immunoassay for concurrent quantitation of C reactive protein (CRP) and tumor necrosis factor-alpha (TNF-alpha) for monitoring inflammatory response. To achieve necessary pore-size control at the gel discontinuity, an optimized mask-based fabrication protocol is introduced. The fabrication approach improves electrophoretic separations using the discontinuous separation gels by eliminating two confounding phenomena: (1) smaller than desired pores at the discontinuity which result in undesired physical exclusion of large-species and (2) an associated transition from small to large pores aft of the interface which acts to "destack" analyte bands during the separation. With the use of the optimized discontinuous separation gels, both assays were linear and quantitative over a two-log detection range, with a lower limit of detection of 11 ng/mL for CRP and 40 ng/mL for TNF-alpha. An optimal single-point detector location was identified by balancing the separation resolution and assay duration constraints. The ultrashort separation distance electrophoretic assays developed here provide flexibility in chip and instrument design by relaxing electrical potential requirements and expanding the possibilities for assay multiplexing, therefore addressing important design considerations when developing field-portable diagnostic assays for near-patient environments.

Clinically relevant advances in on-chip affinity-based electrophoresis and electrochromatography.

Clinical and point-of-care disease diagnostics promise to play an important role in personalized medicine, new approaches to global health, and health monitoring. Emerging instrument platforms based on lab-on-a-chip technology can confer performance advantages successfully exploited in electrophoresis and electrochromatography to affinity-based electrokinetic separations. This review surveys lab-on-a-chip diagnostic developments in affinity-based electrokinetic separations for quantitation of proteins, integration of preparatory functions needed for subsequent analysis of diverse biological samples, and initial forays into multiplexed analyses. The technologies detailed here underpin new clinical and point-of-care diagnostic strategies. The techniques and devices promise to advance translation of until now laboratory-based sample preparation and analytical assays to near-patient settings.