NGS, SCA, Mass Spec: The Road to Diagnostics Agenda

Badge and Conference Materials Pick-Up

Microfluidic Bioaffinity Assays: Improving Detection and Molecular Profiling of Disease Biomarkers
Yong Zeng, Associate Professor, University of Florida, United States of America

Molecular recognition or bioaffinity interaction is an essential principle underlying a vast array of biotechnologies and applications, such as biosensing and drug delivery.  Here we will discuss our studies on exploring microfluidic principles to advance bioaffinity-based assays for quantitative detection and biochemical characterization of circulating cancer biomarkers.  More specifically, this presentation will be focused on microfluidic engineering of both immunoassays and lectin array to substantially improve the assay performance.  Their applications to detection and glycan profiling of circulating proteins markers and exosomes associated with tumors will be presented to demonstrate the potential use for cancer diagnosis.

Highly-Efficient and Selective Isolation of Circulating Tumor Cells by Using a Wave-herringbone Microfluidic Device
Shunqiang Wang, Research Assistant, Department of Mechanical Engineering & Mechanics, Lehigh University, United States of America

We reported a novel microfluidic device with wave-herringbone structures for isolation of circulating tumor cells. Through a geometry optimization study in our proposed computational model, our device achieved both competitively high capture efficiency and selectivity compared with reported values.

Introductions and Agilent Mass Spec Technology Overview
Michael Scott, Clinical Market Development Manager, Agilent Technologies, Inc., United States of America

High Throughput Monitoring of Small Molecules in Human Plasma
Mohit Jain, Assistant Professor, University Of California San Diego, United States of America

Both internal and external environmental factors are critical modulators of human disease through the introduction of small molecules into circulating plasma. Traditional untargeted LC-MS based approaches for assessing plasma small molecules are analytically time intensive, thereby limiting sample throughput. We will introduce new approaches for high throughput measure of small molecule metabolites in human plasma using automated in-line SPE with direct infusion mass spectrometry. We apply these tools to population scale cohorts and will demonstrate the association of small molecules with human disease.

Driving Precision Genomics with Complementary High-Resolution Technologies
David Weiss, Field Application Scientist, Agilent Technologies Inc, United States of America

An Integrated Process for Identification, Retrieval, and Single-Cell Genomic Analysis of Circulating Tumor and Fetal Cells
Eric Kaldjian, Chief Medical Officer, RareCyte, United States of America

Genomic analysis of single cells has been enabled by methods allowing amplification of extremely small amounts of nucleic acid.  RareCyte has developed AccuCyte® - CyteFinder®, an integrated technology platform for highly sensitive visual identification and retrieval of rare cells in blood.  The AccuCyte kit comprehensively collects the nucleated cell fraction of the blood and transfers it to a microscope slide compatible with automated immunostaining and other slide-based tissue tests.  CyteFinder is a highly precise digital scanning fluorescence microscope with image analysis system for multi-parameter visual characterization and mechanical isolation of single cells for genomic analysis for research. Investigational genomic analyses for circulating tumor and fetal cells will be presented.

Close of Agilent Symposium

Conference Registration, Coffee, and Breakfast Pastries in the Exhibit Hall. Exhibit Hall Opens.

Integrated Lens-Free Imaging Technologies Enabling High-Throughput Label-Free Single-Cell Analysis
Richard Stahl, Senior Researcher, IMEC VZW, Belgium

Imagine an inexpensive, portable and label-free (blood) cell analyzer that can be used in emergency or resource-limited settings. Our goal is to use the versatility and scalability of semiconductor technologies to develop such a high-throughput single-cell analyzer using miniaturized lens-free microscopes integrated into a self-contained single-chip system. While our previously reported results confirm that such devices can reach sufficient performance, a number of challenges needs to be addressed in the imaging, micro-fluidics as well as photonic sub-systems. Here we give an overview of the system design, report the progress in development and integration of the individual hardware components, and showcase the system demonstrators with integrated photonics and micro-fluidics. We also report the advances on image reconstruction and cell classification algorithms and discuss the latest results from experiments on analyzing and sorting subtypes of white blood cells with our cell-sorting devices.

Coffee Break, View Posters, Visit Exhibitors and Networking

Tracking Development and Aging of Individual Bacteria with Microfluidic Devices
Stephen Jacobson, Professor, Indiana University, United States of America

Analysis of single cells provides powerful insight into biological processes that are often missed when a population of cells is studied as an ensemble. We are developing microfluidic-based approaches coupled with optical microscopy to track individual bacteria and to improve the temporal and spatial resolution of single-cell measurements. To streamline our experiments, we designed and tested a microfluidic “baby machine,” which automates the culture, synchronization, and analysis steps. This microfluidic device is used to produce synchronized populations of cells and monitor adhesion of individual bacteria to surfaces. To study development and aging in bacteria, we integrated nanochannel arrays into the microfluidic platform that physically trap bacteria. With these nanochannel arrays, we are able to study a number of bacteria strains and observe them over multiple generations. Consequently, we are able to determine rates of cell growth and division, monitor accumulation of cellular damage, and observe epigenetic effects.

Networking Lunch, Visit Exhibitors and View Posters

Technology Spotlight:
Single-Cell Analysis: Guiding the Future of Early Detection, Prognosis, and Course of Treatment for Disease
Naveen Ramalingam, Scientist II, Fluidigm Corporation

A number of breakthrough technologies in single-cell analysis are paving the way for increasing the sensitivity & reproducibility of diagnostic tests, while simultaneously reducing the required labor and turnaround time for results. In this session, we will discuss some of these new breakthroughs and their impact on the community.

Carbon Nanopipettes (CNPs) for Automated Microinjection and the Study of Sub-cellular tRNA Dynamics
Haim Bau, Professor, University Of Pennsylvania, United States of America

We describe the use of carbon nanopipettes (CNPs) for microinjection into individual cells.  The CNP consists of a pulled-quartz micropipette with a thin layer of carbon deposited along its entire interior surface. The tip of the CNP is etched to expose a carbon pipe that varies in diameter from tens to hundreds of nm. The bore of the CNP facilitates microinjection, while the carbon film enables electrical measurements. We utilize the carbon film for impedimetric detection of cytoplasmic and nuclear penetration to trigger injection. We use the CNPs in a custom-developed, automated injection system.  As a demonstration of an application, we describe microinjection of fluorescently-labeled tRNA to monitor subcellular tRNA dynamics in real time.

Coffee Break, View Posters, Visit Exhibitors and Networking

Single Cell Pipette Assisted Cancer Cell Genome Analysis
Lidong Qin, Professor and CPRIT Scholar, Houston Methodist Research Institute, United States of America

There is increasing evidence that solid tumors are likely comprised of many subpopulations of cells with distinct genotypes and phenotypes, which is a phenomenon termed intratumor heterogeneity. Such heterogeneity becomes a major obstacle to effective cancer treatment and personalized medicine. Because of this inherent heterogeneity, data collected from cancer cell population-averaged assays likely hides valuable but rare events such as dramatic variations in gene expression at the single cell level. Therefore, understanding cellular heterogeneity from cancer biospecimens, especially in the study of phenotype-genotype correlation, will facilitate identification of new cell subsets, and assist in cancer prevention, diagnosis, and therapy. In this proposal, we focus on developing a high throughput approach for single-cell isolation based on cells’ capability in generating protrusions. To be able to retrieve the desired single adherent cell, a Protrusion Analysis Chip (PAC) is proposed, in which the single cell is captured by a single hook and physically isolated by a barrier. The PAC is rapid, operationally simple, highly efficient, and requires low-volume sample introduction. After adhesion and spreading, cell phenotype is identified microscopically and then the desired cell is retrieved for genotype analysis. The proposed technology developments and study plans may potentially strengthen the understanding of the relationship between phenotype and genotype at the single cell level.

Microfluidics for Cell Encapsulation
Richard Gray, Regional Director, Blacktrace, Inc., United Kingdom

Microfluidic methods have recently been used to encapsulate thousands of single cells, allowing massively parallel and rapid processing whilst requiring small samples and relatively simple implementation.  Applications recently reported in major journals include high throughput generation of single cell RNA transcriptomes, profiling natively linked T-cell receptors, isolating monoclonal antibodies from blood samples, and directed evolution by library sorting. This presentation will review the devices and systems required and illustrate typical protocols, encouraging wider adoption by scientists.

Fabrication of Polymer Nanofluidic Lab-on-Chip Devices for DNA Manipulation and Large Scale Sequencing
Jeroen A. van Kan, Professor, Centre for Ion Beam Applications, Physics Department, National University of Singapore, Singapore

A new 3D-nanolithographic technique which can produce smooth 3D-nanostructures, will be presented. This technique is used to produce masters for PDMS replication of nanofluidics LOC devices down to 60 nm. These LOC devices are extensively used for DNA single molecule analysis. Here I will give an update on the progress of the single molecule study, eg compaction studies and large scale sequencing. Finally thermal nano imprint lithography will be evaluated for the fabrication of PMMA nanofluidics LOC devices.

Single Cell Random Access Memory
Benjamin Yellen, Associate Professor, Biomedical Engineering, Mechanical Engineering and Materials Science, Duke University, United States of America

The ability to manipulate small fluid droplets, colloidal particles and single cells with the precision and parallelization of modern-day computer hardware has profound applications for biochemical detection, gene sequencing, chemical synthesis and highly parallel analysis of single cells. Drawing inspiration from general circuit theory and magnetic bubble technology, we have recently demonstrated a class of integrated circuits for executing sequential and parallel, timed operations on an ensemble of single particles and cells. The integrated circuits are constructed from lithographically defined, overlaid patterns of magnetic film and current lines. The magnetic patterns passively control particles similar to electrical conductors, diodes and capacitors. The current lines actively switch particles between different tracks similar to gated electrical transistors. When combined into arrays and driven by a rotating magnetic field clock, these integrated circuits have general multiplexing properties and enable the precise control of magnetizable objects.  The presentation will focus on our recent progress on optimizing these integrated circuits to manipulate magnetically labeled CD4+ T cells, including recent data on single cell switches, cell velocities on chip, as well as the ability of non-fouling surface coatings to resist non-specific adhesion between the cells and chip surface.

Cocktail Reception in the Exhibit Hall: Visit the Exhibitors, View Posters and Network with Your Colleagues. Beers, Wines, and Appetizers Served

Close of Day 2 of the Conference

Morning Coffee, Breakfast Pastries, and Networking in the Exhibit Hall

Technology Spotlight:
Advances in Microscopy-Based Single Cell Isolation
Chris Wetzel, Director of Sales and Marketing, MMI Microscope-based Single Cell Isolation

Rare Cell Characterization
Mark Connelly, Chief Industrial Operations and R&D Officer, U.S., Menarini Silicon Biosystems, Inc., United States of America

Tumors spreading via a patient’s vasculature, is a well-established and simple concept. The biology that drives it however, is complex and dynamic. In order to shift cancer treatment regimens from tumor-reactive to tumor-proactive we must develop increasingly sophisticated tools for analyzing circulating tumor materials, properly dissecting the underlying biological complexity, and intercepting the disease earlier in the transformation process. We will present the use of an emerging microfluidic technology whose negative depletion technology demonstrates the ability to enrich and purify essentially all populations of circulating tumor cells, tumor related cells, as well as harvest plasma for cfDNA and other markers.  Molecular and phenotypic analysis on enriched products will illustrate the ability of the platform to be used for research on non-epithelial tumors and to investigate early disease interception.

Coffee Break, View Posters, Visit Exhibitors and Networking

Integration of Genomic and Proteomic Approaches through the NantHealth Operating System
Frank Ong, Executive Director, Medical Affairs and Clinical Development, NantHealth, United States of America

NantHealth, a member of the NantWorks ecosystem of companies founded and led by Dr. Patrick Soon-Shiong, is a transformational healthcare company converging biomolecular medicine and bioinformatics with technology services to transcend the traditional barriers of today’s healthcare system through a single integrated clinical platform. NantHealth works to transform clinical delivery with actionable clinical intelligence at the moment of decision, enabling clinical discovery through real-time machine learning systems. The NantHealth Operating System (NantOS) platform empowers physicians, patients, and payers to coordinate best care, monitor outcomes and control cost in real time. This is the first operating system of its kind in healthcare, enabling real time data capture from multiple disparate data sources, and is based on supply chain principles and grid service oriented architecture, integrating the knowledge base with the delivery system and the payment system, enabling 21st century coordinated care at a lower cost, with value-based population health management at a single patient level and at the population at large.

The Concordance of RNA-seq and Microarray is Dependent on Chemical Treatment and Transcript Abundance
Charles Wang, Director & Professor, Loma Linda University, United States of America

Next-generation sequencing technologies have revolutionized the genomic research and allow the genome and transcriptome of any organism to be explored without a priori assumptions and with unprecedented throughput. However, the concordance of RNA-sequencing (RNA-seq) with microarrays for genome-wide analysis of differential gene expression has not been rigorously assessed using a range of chemical treatment conditions. Here we use a comprehensive study design to generate Illumina RNA-seq and Affymetrix microarray data from the same liver samples of rats exposed to varying degrees of perturbation by 27 chemicals representing multiple modes of action (MOAs). The cross-platform concordance in terms of differentially expressed genes (DEGs) or enriched pathways is linearly correlated with treatment effect size (R2˜0.8). Furthermore, the concordance is also affected by transcript abundance and biological complexity of the MOA. RNA-seq outperforms microarray (93% versus 75%) in DEG verification as assessed by quantitative RT-PCR, with the gain mainly due to its improved accuracy for low-abundance transcripts.

Networking Lunch, Visit the Exhibitors, and Poster Viewing

Technology Spotlight:
Acoustic Liquid Handling for Genomics Applications
Austin Swafford, Field Application Scientist, Labcyte Inc

The Labcyte Echo® liquid handler revolutionizes liquid transfer by using acoustic energy to eject fluids. Echo liquid handlers are contact-free – no tips or nozzles are used as nanoliter-sized droplets of the fluid move directly from source plate to destination plate. Eliminating tips reduces costs, minimizes waste and eliminates carry-over cross contamination. Dispensing sub-microliter volumes further enables assay miniaturization to provide additional cost savings and to conserve precious samples and reagents. The technology behind Echo liquid handlers and the Genomics application areas where the systems are currently in use will be reviewed in the presentation.

Metabolomics: The Missing Link in Precision Medicine
John Ryals, President And CEO, Metabolon Inc, United States of America
Shaun Lonergan, Vice President/General Manager, Metabolon Inc, United States of America

Metabolon has developed a platform technology capitalizing on advances in mass spectrometry, proprietary software and database analysis to provide unprecedented insight into biochemical pathways. Along with information provided by whole genome or exome sequencing technologies, metabolite data provides ontology for determining genome wide associations.  This insight is critical to determining penetrance of a gene determinant of interest and relating it to health status.   Because metabolites are more closely linked to actionable information, metabolomics can provide clinicians with more insight to the patient’s health status.  With or without genetic information, metabolomics will play a big role in the future of precision medicine.

Coffee Break, Networking, Visit the Exhibitors and Poster Viewing

Bioanalytical Sample Preparation Strategies Prior to LC-MS/MS Analysis of Drugs and Biomarkers
Vincenzo Pucci, Principal Scientist, Preclinical Bioanalytical Group, Merck Research Laboratories, United States of America

An essential component of the drug-discovery process is the determination of drug and biomarker concentrations in biological samples which yields the data necessary to understand the pharmacokinetics and pharmacodynamic, respectively. To obtain these data, each sample of fluid obtained from an in vitro or in vivo study is subjected to sample preparation, chromatographic separation, and analytical detection. Sample preparation is an important and necessary step in the overall analytical process, because most analytical instruments cannot accept the sample matrix directly. Despite the tremendous advances in liquid chromatography mass spectrometry (LC-MS) and LC tandem mass spectrometry (LC-MS/MS) detection systems in recent times, sample preparation still represents the most challenging part of the bioanalytical workflow. The removal of unwanted endogenous matrix components (e.g., protein, salts, etc.) from the sample must be rapid and as complete as possible to avoid the introduction of interferences upon analysis. Many different techniques are available to a drug discovery scientist performing bioanalytical sample preparation. The focus of this presentation is to introduce the goals for sample preparation and the many choices available to the drug-discovery scientist.

Automated, Multiplexed Quantitation of Proteins; Facilitating Mass Spectrometric Assays in Research and in the Clinic
Selena Larkin, Vice President, Marketing and Sales, SISCAPA/Omicia, United States of America

Proteomics workflows often lack the necessary reproducibility required for clinical applications. This is in part due to the complex nature of these workflows, which typically start with enzymatic digestion (e.g. trypsin) of complex specimens (e.g. human blood) followed by multiple fractionation steps prior to analysis by a mass spectrometer. Therefore, in order to comply with the clinical specifications for total analytical error, a simple and reproducible workflow is needed. Here we report the development of a generic, highly reproducible tryptic digestion method for complex protein mixtures. This digestion approach is further complemented with the use of the SISCAPA technology (sequence-specific capture of target peptides and corresponding stable isotope labeled standards using high-affinity anti-peptide antibodies from digested specimens). The result is an ‘addition-only’ or homogeneous approach that can be easily implemented on a variety of automated liquid handling platforms for precise, robust and sensitive measurement of peptide analytes. The addition-only strategy allows for sequential addition of samples and reagents in a 96-well format and eliminates the need for separative techniques such as filtration, solid phase extraction or centrifugation. Furthermore, the SISCAPA enrichment of analytes creates eluted samples that can be analyzed by mass spectrometry with minimal liquid chromatography. We have implemented this workflow on a Bravo Liquid Handling Robot (Agilent Technologies, CA) for liquid specimens (e.g. whole blood, plasma/serum) and dried blood spots (DBS). In collaboration with Agilent Technologies, we have also designed an intuitive user interface that further simplifies the workflow from an operational perspective.

Close of Day 3 of the Conference