41st Annual Oak Ridge ConferenceFrontiers in Clinical Diagnostics
April 16 & 17, 2009 – Baltimore, Maryland
Poster 1Microfluidic chip detection of HLA-B*5701. Avula R, O Kane DJ. Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN.
Introduction: Abacavir is an effective drug used for the management of HIV. However, abacavir has been found to cause a drug hypersensitivity reaction in 5-8% of patients initiating therapy. Clinical symptoms of hypersensitivity which include fever, rash, gastrointestinal symptoms, lethargy, and malaise, usually occurs within 6 weeks of commencing abacavir therapy and can be severe and life threatening. Several studies have demonstrated a clinical association between carriage of a particular genetic allele of the major histocompatibility complex, HLA-B*5701, and increased risk of hypersensitivity. The association is strongest in individuals of Northern European descent but weaker in other populations. The FDA recently recommended that people have genetic testing for HLA-B*5701 before taking Abacavir. Standard serological approaches used to detect HLA-B*57 lack specificity, as commercially available monoclonal antibodies cross-react with HLA-B57 and HLA-B58 subtypes. Serological methods require intact cells and may also result in false-negative results if HLA-class I molecules are down-regulated during infections. High resolution HLA typing and HLA-sequencing methods are labor intensive and not cost effective. Here we validated a rapid HLA-B*5701 molecular detection method on Agilent 2100 Bioanalyzer. Method: This molecular detection method consists of multiplexed PCR using sequence –specific primers (SSP). This system allows rapid HLA*5701 low resolution PCR typing using a primer mix containing four specific primer that can be used to discriminate HLA-B*5701 from the related B57 subtypes. Following PCR the HLA-B*5701 allele is detected by size determination of the products using Agilent 2100 Bioanalyzer. Microfluidic Lab-on a Chip technology applies the principle of electrophoresis to network of channels and wells etched into polymer chips, is easy to use, requires low sample volume and analyses 12 samples in 30 minutes. The samples used for validation were verified by bi-directional DNA sequencing of HLA-B exon 3.Results: The assay was validated using 42 unknown DNA samples extracted by 2 different methods (Qiagen EZ-1 extraction kit and Quickgene-810 extraction method) and also 35 DNA samples purchased from the Coriell Institute. Among the Coriell DNA, 20 were Caucasians and the rest were Chinese, Japanese, Southeast Asians and Hispanic. We found 10 samples positive for HLA-B*5701. In addition, positive controls for *5701 and *5703 (which has a positive band for only 57 antigen) purchased from UCLA Immunogenetics Center were used in validation of the assay. A subset of both *5701 positive and negative samples were sequence verified by sequencing exon 3 region of HLA-B gene. The sequences under the primers were verified in this sequencing and there was 100% concordance with the Agilent results.Conclusion: This assay is simple, rapid, and cost effective method for detecting HLA-B*5701. Detection of PCR products on Agilent Bioanalyzer avoids the labor intensive procedure of running agarose gels. This method has the sensitivity and specificity to differentiate commonly occurring HLA-B57 subtypes.
Poster 2Magnetizable proteins with antibody like binding properties. Dehal PK1, Livingston CF1, Geekie C1, Pritchard DJ1. 1Research and Development. Axis-Shield Diagnostics Ltd, The Technology Park, Luna Place, Dundee, DD2 1XA Scotland, UK.
The objective of this study was to design and produce inherently magnetizable proteins with binding properties similar to those of antibodies. These magnetizable proteins would provide an alternative to traditional inorganic paramagnetic particles. The use of inorganic paramagnetic micro or nano particles to isolate molecules or cells from complex media is well established in a number of fields including clinical chemistry. Typically, inorganic particles are manufactured and subsequently coated with a biological molecule which confers specific biorecognition capability. Incubation of particles with the sample and exposure to a magnetic field isolates the species of interest. We designed, produced and assessed magnetized fusion proteins consisting of the antigen binding portion of a monoclonal antibody (single chain Fv; scFv) fused to subunits of the iron binding protein ferritin. The fusion protein subunits expressed in E. coli assembled to form a fusion protein consisting of a ferritin sphere with scFvs on the surface. Fusion proteins were made magnetizable by introducing a paramagnetic iron core. Images generated using transmission electron microscopy demonstrated a mean molecular diameter of 19.5nm (closely matching the predicted diameter of approximately 20nm based upon reported sizes of ferritin and scFv) and demonstrated aggregations of iron within the central cavity. The resultant fusion protein was shown to be magnetizable and it could be selectively retained in magnetic fields. The fusion protein was capable of binding target antigens with high affinity (e.g. Kd 65nM for an anti-fibronectin:ferritin fusion protein when tested with human fibronectin). Additionally, as a model system, it was demonstrated that the fusion proteins could selectively bind platelets from whole blood and retain them in a magnetic field within a fluidic device. The use of these "organic" magnetizable particles may prove to be an attractive alternative to traditional inorganic particles as the fusion proteins possess a number of theoretical advantages.
Figure 1. Transmission electron micrograph of the magnetized scFv:ferritin molecules. The average diameter of the molecules is 19.5nm. Photograph b, is a close-up of one of the molecules. Aggregation of iron can be seen within the core.
Poster 3Rapid, inexpensive, accurate, and user friendly DNA detection for diverse settings. Lipscomb JH 1, Krider H 1, Raffauf, Sr. R1, Robert Bernstine 2, Albrecht K 3. 1X-Bar Diagnostic Systems, Inc., Mendenhall, PA., 2Intuition Design, Inc., 3Department of Genetics and Genomics, Boston University School of Medicine, Boston, MA.
Many field based operations such as In vitro diagnostics in diverse settings, Insect testing, environmental testing, water analysis, and food testing could benefit from DNA testing, but have excluded themselves from considering it because of the cost, and difficulty associated with current transportable testing equipment.The "X-bar I, All-in-One" is a fully automated, portable battery operated biological agent detection system. It consist of a rotary thermal cycler processing station for sample preparation and amplification, a reading station for DNA detection; A CPU for controlling system functions, solar panel battery refresh modules for recharging the battery, an LCD digital monitor, a USB interconnection for printer and remote laboratory computer information management system, and virtual keyboard for operator interface and to prevent liquid spills from shorting out the keyboard. All this in an easy to carry laptop-carrying case (similar to a laptop computer). This novel instrument is designed to enable convenient PCR testing in rugged environments, and a thru-put of 108 test per hour.PCR testing is well established in food testing laboratories using bench top equipment to detect specific sequences of DNA trace samples. Yet most of the laboratories are struggling with the issue of cluttered bench top space due to current bulky DNA processing and testing equipment, along with a full size computer, monitor, keyboard and printer. They also have the issue of having to conduct the three different DNA processing steps in separate rooms because of potential contamination of other samples with nucleic acid.Other features of this interesting device include:-Positive sample identification-5-20 minutes to reaction endpoint using fluorescent tag chemistry-Controls to prevent false positives and false negatives-throughput of 108 samples per hour-Potential for DNA amplification with detection monitored each cycleBenefits:-Able to operate with ease in rugged field environments-Prompt results on the scene-Expandable for higher sample counts per hour (up to 360 samples per hour if needed).-Very affordable instrument and test cost-Closed tube to prevent cross contamination-Can be operated in any country without power conversion devices.
Poster 4New fixation technology for simultaneous preservation of morphology and nucleic acids in tissue. Grölz D1, Lenz C2, Dettmann N2, Hilker M2, Tränert E2, Oelmüller U1, Rainen L1. 1PreAnalytiX GmbH, Hombrechtikon, CH, and 2QIAGEN GmbH, Hombrechtikon, CH.
Introduction / Background: Current tissue fixation methods used in traditional histology are of limited use for molecular analysis. Fixatives which contain formaldehyde cross-link biomolecules and destroy or modify nucleic acids and proteins during fixation. Fixation of tissue in liquid nitrogen, while it preserves RNA and DNA, ultimately leads to disruption of morphological structures. We have developed a new method, the PAXgeneTM Tissue System, which simultaneously preserves tissue morphology and stabilizes nucleic acids. Materials and Methods: Tissue specimens from rat kidney, spleen, liver and intestine were treated with either neutral buffered formalin (NBF), PAXgene Tissue Fix, or frozen in liquid nitrogen. After 2-4 hours at room temperature, PAXgene Tissue fixed specimens were transferred into PAXgene Tissue Stabilizer. PAXgene stabilized samples were then stored at 2-8°C, 18-22°C, and -20°C for up to four weeks. Nucleic acid isolation was performed with PAXgene Tissue Kits. Nucleic acids were purified either directly from 10mg of PAXgene treated tissue or from PAXgene treated tissue which had been embedded in paraffin. The integrity of RNA was determined on the Agilent 2100 Bioanalyzer and that of DNA by gel electrophoresis. Real-time qRT-PCR or qPCR assays were performed on the resulting RNA or DNA. Tissue morphology was investigated by H and E staining of 4µm sections of paraffin-embedded samples. An immunohistochemistry (IHC) assay was performed using antibodies directed against the Ki-67 antigen.Results: Tissue specimens fixed with PAXgene Tissue Fix were stable in PAXgene Tissue Stabilizer at room temperatures (18–22°C) for up to 7 days and at lower temperatures (2-8°C and -20°C) for at least 4 weeks. PAXgene stabilized samples could be embedded in paraffin and used for histological studies even after storage at -20°C. H and E staining patterns and staining intensity in the IHC assay were comparable to those seen with formalin fixed tissue. High molecular weight RNA and DNA could be isolated from the stabilized tissue before or after paraffin embedding. Integrity, yield and performance in qRT-PCR of RNA were comparable to that in tissue frozen in liquid nitrogen. RIN values from sections of PAXgene treated, paraffin-embedded tissue ranged from 6 to 8. All RNA samples allowed successful RT-PCR amplification of amplicons up to 1065bp.Conclusion: The PAXgene Tissue System provides a novel method for stabilization of molecular content and preservation of morphology from the same specimen thus enabling both molecular and traditional pathology testing from the same specimen. The PAXgene Tissue System is for research use only, not for use in diagnostic procedures. Poster 5Rapid visualization of genomic DNA from clinical specimens. Klonoski, J1, Ward, D2, Jenison, R1. 1Great Basin Scientific 2400 Trade Center Ave., Longmont, CO, 80503, 2Nevada Cancer Institute, Las Vegas, NV.
Current methods for rapid detection of nucleic acids in clinical samples are generally limited to target amplification methods with fluorescence-based detection of amplified products in real time. However, due to require labor intensive and difficult sample preparation methods and the expensive instrumentation necessary for amplification and detection of target sequences, their use in resource limited settings is untenable. Herein we describe a simple, rapid method for the direct detection of genomic DNA sequences, termed AMPED (Acetate Modified Polymer Enhanced Detection). AMPED utilizes a thin film biosensor, containing immobilized DNA probes, in conjunction with an inexpensive dextran polymer appended with multiple biotin reporter molecules. The biotinylated polymer is treated to limit non-specific interactions allowing for analytical limits of sensitivity of approximately 100 zeptomoles in a 100 L sample volume. We applied AMPED to the detection of MRSA directly from positive blood culture bottles. Crude lysates from a blood culture aliquot containing Staphylococcal genomic DNA were combined with complementary biotin-labeled reporter probes and hybridized to probes on the biosensor surface. Streptavidin was then bound to the surface-immobilized Staphylococcal genomic DNA/biotinylated probe complex to serve as a bridge to a biotinylated dextran polymer bound. Upon subsequent binding to streptavidin/horseradish peroxidase (SA-HRP), the resulting complex transduces formation of thin films, by deposition of precipitable products of substrate turnover by HRP, which are permanent and clearly visible to the naked eye. In time course studies as little as 5 minutes of hybridization of a crude lysate of blood culture containing Staphylococcal genomic DNA was sufficient to produce a visible signal. Based on this data we were able to configure a test that can detect MRSA in 25 minutes from blood culture bottles; the test was validated with 50 clinical specimens of blood culture bottles determined to be gram positive cocci in clusters and showed complete concordance with standard microbiological methods. The AMPED method, requiring no instrumentation other than a simple heater, may be adapted for detection of DNA sequences from other clinical specimens as well in a low cost, easy-to-perform format amenable to testing in low resource settings such as smaller hospitals or even the point-of-care.
Poster 6Development of a point-of-care measurement system utilizing fluidic control technology. Saiki H, Sugimoto H, Okada K, Kouyama Y. Panasonic Shikoku Electronics Co., Ltd., Ehime, Japan.
Medical practices have great need for devices that will aid in rapid diagnosis. These devices should be simple to operate, and ideally should match the accuracy of results that could be obtained in a clinical laboratory. We have developed unique fluidic control technology that controls centrifugal force, capillary force, gravity, and inertia and incorporated this technology into a POC measurement system capable of meeting these stated needs with just a 10uL finger-stick blood sample.It is difficult to mix quickly and uniformly in the micro area using well known methods such as diffusion phenomenon or centrifugal force. This is due to differences in sample volume and specific gravity between the blood sample and diluents.To overcome this problem, we developed two important technologies. The first is a mixing technology that forcefully initiates a turbulent flow capable of mixing swiftly and uniformly. The second is a fluid transfer technology capable of controlling any fluidic sequence of an assay reaction.With respect to the mixing technology, we came to the conclusion that utilizing inertial force is desirable for rapid and uniform mixing. Specifically, by impressing an inertial force via waggle action, turbulent flow and accelerated mixing were initiated. As a result, we were able to achieve a uniform mixture in a matter of seconds.With respect to the fluid transfer technology, we found that by setting a gap between the outlet port and the fluid level of the chamber and initiating oscillations at a prescribed location with a tilted rotating shaft, we were able to transfer the fluid at any timing in the assay sequence.The Point-of-Care measurement system we developed using our unique fluidic technology, has demonstrated excellent accuracy for our first target markers of Lipid Profile (TG, TC, HDL, LDL) and HbA1c.
Poster 7The detection of HIV-1 proviral DNA in less than 20 minutes: The potential for recombinase polymerase amplification technology as a diagnostic tool in resource limited settings. Boyle DS1, Lehman DA2, Overbaugh J2, Piepenburg O3, Armes NA3, Singhal M1, Gerlach JL1, Weigl BH1. 1Program for Appropriate Technology in Health (PATH), Seattle, WA, 2Fred Hutchinson Cancer Research Center, Seattle, WA, and 3TwistDX Ltd, Cambridgeshire, UK.
Abstract: In Sub-Saharan Africa more than 50% of children infected with HIV die before reaching 2 years of age. Assays available for infant HIV diagnosis require nucleic acid amplification of either proviral DNA or viral RNA. The complexity of specimen collection and stability, sample preparation, storage of test reagents, as well as sophisticated equipment requires highly trained personnel and fully equipped laboratories. Therefore, screening for infant HIV is typically limited to top-tier laboratories located in urban areas. This significantly adds to the cost per test and often creates a delay in reporting of results or renders testing unfeasible due to the distance between clients and the laboratory. To avoid delaying treatment of children infected with HIV, there is an urgent need for an assay to detect proviral HIV DNA that can be completed at or closer to the point of care.In this study we investigated the use of an isothermal DNA amplification method, Recombinase Polymerase Amplification (RPA), to detect HIV proviral DNA. Isothermal amplification technologies are attractive candidates for point-of-collection testing because amplification occurs at a lower constant temperature, reducing the necessity of high power requirements for cycling of heating/cooling and a complex infrastructure to operate in. Two RPA assays were designed to detect pol from HIV-1BRU proviral DNA via real time detection of amplification using a small and comparatively simple reactor/fluorescence reader and by amplicon detection via lateral flow strips, a method of detection requiring no instrumentation. The complete reagent mixes for both assays were supplied lyophilized, reducing reaction set up to only rehydration and the addition of sample. Sensitivity testing was performed on human DNA spiked with quantitated HIV-1 proviral DNA (38 to 1 copies/reaction). Preliminary testing of 79 specimens assayed with the real-time method gave 100% (14/14) detection of 38 copies/reaction and samples with 19 copies were detected at 86% (12/14). Sensitivity was reduced in reactions with lower ACH2 copies with detection rates of 50% (2/4), 25% (5/15) and 4% (1/25) for 10, 3, and 1 copies of ACH2 respectively. With the lateral flow strips fewer samples were screened. The sensitivity data was similar to the real time data with 100% detection of 38 (4/4), 19 (4/4) and 3 (1/1) copies and 50% detection of 1 copy (2/4). All human DNA-only control specimens tested negative. Completed run times and the detection of amplicons were achieved within 20 and 30 minutes from the start of amplification with the real-time and lateral flow assays respectively. This is significantly faster than current nucleic acid amplification strategies. The RPA assays have a short reaction run time in addition to simple reaction set-up, high sensitivity and specificity. The low power requirement, ease of use, and relative low cost of the RPA real time instrument and an alternative lateral flow assay that needs only a basic heating block suggests that RPA is an emerging technology with great potential for the diagnostic screening of infants for HIV at point-of-care locations in low-resource settings.
Poster 8Point-of-care serology system for HIV and opportunistic infection screening and diagnosis. Lochhead MJ1, Todorof K1, Delaney M1, Heil JR1, Ives J1, Zhang X2, Reed S2, Schooley RT2, Myatt CJ1. 1mBio Diagnostics/Precision Photonics Corporation, Boulder, CO, and 2University of California, San Diego, CA.
Diagnosis and management of HIV-infected individuals continues to be a major public health priority around the globe. Although the emergence of rapid tests for HIV screening over the past decade has greatly improved disease management for individuals and across populations, most current rapid tests are limited to providing qualitative detection HIV-1 or HIV-1&2 antibodies. Management of HIV infected individuals would be greatly improved with actionable co-infection information at the time of diagnosis or at the time of initiation of antiretroviral therapy. Current standard-of-care includes determinations of related infections such as hepatitis C virus (HCV), hepatitis B virus (HBV), T. gondii, T. pallidum, and cytomegalovirus (e.g., 2008 Recommendations of the International AIDS Society – USA Panel, Hammer, et al., JAMA 2008, 300(5), 555-570; and CDC/HIV/IDSA Guidelines in Benson, et al. MMWR 2004, 53, RR-15, 1-112). These multiple diagnoses typically require extensive use of serological diagnostic tools, often in diverse test formats. This often renders co-infection diagnosis cost-prohibitive, particularly in high HIV prevalence, resource-limited settings.mBio Diagnostics is addressing the critical need for co-infection diagnostic tools through development of a fluorescence immunoassay system that provides serology data for HIV and a panel of opportunistic co-infections. The system uses a 20 microliter sample in an instrumented package with costs and workflow conceptually similar to current rapid tests. The system combines a simple instrument with disposable test cartridges. The instrument uses a novel waveguide illumination approach combined with consumer electronics components, enabling a low-power, extremely low cost manufactured instrument. The disposable cartridge integrates a low density microarray with a fluidic chamber that provides an assay analogous to lateral flow devices. The cartridge has been designed for manufacture with conventional fabrication approaches. Cartridge components, including the optical waveguide, are injection molded plastic. Additional advantages of the system relative to current rapid tests include automated analysis, quantitative output, and potential integration with lab information systems.Clinical sample data are presented for an HIV / hepatitis C virus (HCV) serology panel. The HIV/HCV cartridge includes an array of three HIV antigens (gp41, gp120, and p24), two HCV antigens (HCV core and c33c), as well as in-array sample and procedural control spots. Data are presented for 40 sera samples collected from a cohort of high-prevalence HIV/HCV individuals in San Diego, CA, and an additional archived set of 30 characterized plasma samples. Based on preliminary positive/negative call algorithms developed using these 70 clinical samples, initial analysis shows 100% specificity for HIV and HCV, 95% sensitivity for HIV, and 96% sensitivity for HCV. In addition to this clinical dataset, results will be presented for field trials performed with prototype instruments operating in clinical laboratories at the University of California, San Diego Medical Center. Panel expansion to include HBV, T. gondii, and T. pallidum will also be discussed.
Poster 9Integrating microfluidics and lens-less imaging for point-of-care testing. Moon SJ1, Keles HO1, Khademhosseini A2,3, Kuritzkes D2, Demirci U1, 3. 1Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Center for Bioengineering, Brigham and Women's Hospital, Harvard Medical School, 2Brigham and Women's Hospital, Harvard Medical School, and 3Harvard-MIT Health Sciences and Technology, Cambridge, MA.
Learning Objectives: An integrated platform that merges a microfluidic chip with lens-less imaging to target CD4+ T-lymphocyte counts for point-of-care testing at resource-limited settings. Background: The CD4+ T-lymphocyte count is performed currently 3 ~ 4 times a year in the developed world, and twice a year in the developing world using fluorescent activated cell count and sorting systems (FACS). ART is started for infected persons whose CD4+ T-lymphocyte count is below 200 ~ 350 cells per microliter. There is a need for rapid diagnostic and monitoring systems that are simple-to-use, inexpensive, reliable, and disposable to complement current monitoring methods. Design/Methods: The chips were designed and fabricated simply with a laser cutter instead of using expensive cleanroom equipment. To capture CD4+ T lymphocytes, anti-CD4 antibody was immobilized on one side of the microfluidic chip. These captured cells were detected through an optically clear chip using a charge coupled device (CCD) sensor by lens-less shadow imaging techniques, fig. 1.Results: Figure 2 shows the microfluidic chip capture specificity, capture efficiency, CCD efficiency, and overall platform performance. The chip specificity and efficiency are related to the surface chemistry and shear based mechanical filtration method. The average value of the three blood samples shows 88.8 ± 5% capture specificity for CD4+ cells and 70.2 ± 6.7% chip capture efficiency (n = 9 devices). Such a rapid CD4 count allows fast feed back at the POC, when compared to existing systems (e.g., magnetic beads: 5 ~ 10 test per day) and flowcytometry (30 ~ 50 test per day, including incubation times for fluorescent cell staining). Overall platform performance is important for clinical applications. The mean and the standard deviation of overall platform performance was 83.5 ± 2.44 %. The repeatable performance allows correcting for the length dependent counting bias (in this case divide by the mean value, 0.835). The corrected CD4+ cell count estimate should be clinically acceptable given that the chip capture efficiency is repeatable within 2.44 %. This standard deviation value currently satisfies the clinical need (± 10 % overall count error) with the bias correction approach.Conclusions: We demonstrated a novel method to build a point-of-care device that can be merged with lens-less imaging for rapid automatic cell counting, i.e. CD4 counts. The lens-less CCD imaging platform merged with label-free cell capturing is potentially useful for resource-limited settings, since it eliminates the need for fluorescent imaging; it reduces the time for cell capture, imaging, and counting to a few minutes from hours. The merger of the microchip with the CCD was successful to capture, image and automatically count the CD4+ T-lymphocytes. This integrated system poses a future direction for point-of-care testing especially focusing on global health applications at resource limited settings.
Poster 10Use of peptide nucleic acid probe for homogenous detection of low abundant, amantadine-resistant influenza A virus. Chiou CC, Cheng TL. Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan.
Detection of drug-resistant mutant of pathogens is important for a clinician to choose an appropriate therapeutic strategy. However, to detect such mutants is usually difficult as they might only exist in a low abundance in the population before treatment. In this study, we have developed a novel method combining an anchor probe and a fluorophore-labeled peptide nucleic acid (PNA) probe that covered the mutation hotspot of matrix protein 2 gene of influenza A virus for homogenous detection of Amantadine-resistant virus. PNA is a DNA analog in which the phosphodiester backbone is replaced with an N-(2-aminoethyl)glycine chain. A wild-type specific PNA oligomer can selectively inhibit the amplification of wild-type template but allow the amplification of mutant templates. The fluorophore-labeled PNA was also designed as a sensor probe to reveal the melting curve profile of the PCR products. By the melting curve analysis after PCR amplification, the mutant could be identified through its signature melting peak. On a LightCycler machine, this assay can be finished in a single tube in less than an hour. The detection limit of this assay is 10 copies of mutant viral genome in the existence of 100-1,000 fold excess of wild type genome. Using this method, we have successfully identified 10 mutants from 21 amplifiable, throat swap samples collected from patients before treatment, which is in agreement with the results using traditional methods involving viral culture, PCR, and sequencing. However, we did not find mutant quasi species in the wild type population, indicating that the Amantadine-resistant mutant may not co-exist with the wild-type virus in most patients, or that the mutant exist in an even lower ratio than the detection limit. In summary, we have developed a rapid and sensitive method using PNA probe to enrich and detect low abundant viral mutants. This method may also be useful for the detection of rare drug-resistant mutants in other pathogens and cancer cells.
Poster 11The CD4 initiative: enabling the research and development of a simple, point-of-care assay for CD4 testing in HIV infected individuals in resource limited settings. A. Burshteyn, E. Jachimowicz, G. Spruill, I. Munoz-Antoni, I. Borodowsky, J. Wen and S. D'Costa. Beckman Coulter, Miami, FL.
Introduction*: There are around 40 million people worldwide living with HIV, the majority of who live in resource-limited countries where access to diagnostics essential for therapeutic management of the disease is limited or absent. An example of such an essential diagnostic is CD4 T cell enumeration which is used • as a surrogate marker of disease progression, • to determine the need for initiation of antiretroviral therapy • to evaluate the responsiveness to antiretroviral therapy. The CD4 enumeration assays routinely run in centralized laboratory settings require trained laboratory technicians, expensive equipment and reagents, all of which are severely limited in resource poor countries. With a view to enabling quality disease management and informed therapeutic treatment of HIV in resource limited settings, the CD4 Initiative, at Imperial College, London, funded by the Bill & Melinda Gates Foundation was instituted. The CD4 Initiative awarded funds for the development of an easy, cheap, non-instrument based point of care (POC) CD4 enumeration assay on a competitive basis. Funds awarded to academic, private and public institutions resulted in a unique, multi-partner collaborative and at the same time, competitive approach for the development of the POC assay. The current poster will address the ongoing activities at Beckman Coulter Inc. for the R&D of a non-instrument based, CD4 T cell POC assay meeting the specifications relevant to resource limited settings.Methods: A variety of commercially available and custom-cast absorbent, non-absorbent material and capture antibody combinations were evaluated for the specific capture of intact CD4 + T cells from whole blood.1. A variety of methodologies to deplete monocytes from whole blood were evaluated to limit the impact of CD4+ monocytes on the assay readout2. A variety of detector antibodies and visible readout combinations were assessed to identify the most optimal non-instrument based readout.3. A variety of assay steps impacting time to result were assessed to meet assay throughput specifications.Combinations of above assay components and methodologies were evaluated to enable the semi-quantitative detection of CD4 T cells from whole blood and identify the need for initiation of anti-retroviral therapy in HIV infected individuals using the non-instrument based POC assay.Whole blood obtained from 32 HIV infected individuals were evaluated using the above described POC assay (currently in the feasibility stage) and compared to gold standard flow cytometry based assays. Results: Preliminary data evaluating HIV positive donors determined that the current POC prefeasibility assay has a sensitivity of 72% and specificity of 85% when compared to gold standard flow cytometry.Conclusions*: Studies that began from the concept phase in May 2007 have determined that a non-instrument based POC assay for semi-quantitative enumeration of intact CD4 T cells, the 1st assay of its kind, is indeed feasible. Additional studies to improve the sensitivity, specificity, precision, robustness and ease of use of the current assay to meet the CD4 Initiative specifications are in progress.Acknowledgements: Dr. Hans-Georg Batz, and Dr. Steven Reid at Imperial College, London. Dr. Enrique Rabellino and Dr. Wade Bolton at Beckman Coulter Inc.
Poster 12Reagentless electrochemical biosensors for clinical diagnostics. Georganopoulou D. Ohmx Corporation, Evanston, IL.
Ohmx Corporation is currently developing a reagentless biosensor system using a non-antibody based approach for the electronic detection of biomarkers in clinical samples. Central to Ohmx's proprietary technology is the electrochemical reorganization energy of metal reporter tags attached to capture ligands and anchored as self-assembled monolayers (SAMs) on electrode microarrays. Because Ohmx`s technology is based on electronic detection without reliance on antibodies, the overall cost can be minimal. Each microelectrode can be modified by different capture ligands allowing for multiplexed detection of different biomarkers in one sample. Current advances in the methodology strive to improve the system's sensitivity and detection limit for a variety of applications in clinical diagnostics. Coupling these efforts with miniaturization and improved time-to-answer, the Ohmx sensor aims to become the diagnostic platform of choice for protein detection in the laboratory, doctor's office, at the point-of-care, and in the future, the home.
Sample preparation is an important step for the detection of nucleic acids for many molecular clinical tests. There is an inherent challenge to DNA sample preparation from large volume clinical samples where target is present at a low concentration such as cervical samples in liquid based cytology media. Most solutions offered by the market today involve centrifugation or nonspecific absorption on magnetic beads; however both require steps and apparatus that may decrease assay throughput and generate a complex mixture of cellular components that may negatively affect subsequent applications. QIAGEN's proprietary Hybrid Capture technology offers a unique solution to large volume sample processing that leverages the technology behind the digene hc2 HPV test. This method utilizes bioinformatically-designed short synthetic oligoribonucleotides (synRNA) to specifically hybridize to target DNA. These DNA:RNA hybrids are captured by Hybrid Capture antibodies conjugated to magnetic beads. This method does not involve any centrifugation or transfer steps and generates significantly enriched material to be detected. The feasibility of large volume Hybrid Capture was demonstrated utilizing two standard liquid based cytology media: PreservCyt (Hologic) and SurePath (BD Diagnostics). Adaptations to Hybrid Capture allowed direct capture and detection of 15,000 copies of HPV DNA in 1 mL PC or SP in less than 3 hours. In this case, detection of captured target was performed without target amplification by subjecting captured target to signal amplification provided by Hybrid Capture antibodies. Large volume Hybrid Capture sample preparation is also compatible with two distinct target amplification technologies: PCR and Whole Genome Amplification (WGA). In the same time period, a sensitivity of 100 copies HPV16 target was detected with an input volume of 2 mL PC by Hybrid Capture combined with WGA. The combination of Hybrid Capture sample prep with target amplification offers a viable solution that provides both extreme specificity and sensitivity for clinical assays.
Poster 14Effect of sample flow rate on the sensitivity of an ion channel switch bilayer sensor. Cornell B1, Handy M2, Mangin A-G3, Richards R1. 1Surgical Diagnostics PL., Sydney, Australia, 2 University of Technology, Sydney, Sydney, Australia, and 3Claude Bernard University of Lyon, Lyon, France.
The response to streptavidin in the range 100fM to1pM of a biotinylated gramicidin, ion channel switch (ICS) sensor has been studied at varying capture site densities and sample flow rates. Although streptavidin is reported here, the conclusions also apply to streptavidin-antibody conjugates that form part of an immunoassay. Electrical impedance measurements were made of a 2mm x 2mm test sensor sandwiched within a 0.1mm rectangular flow cell. Ion channel densities of 108cm-2 and capture site densities from 108cm-2 to 1011cm-2 were studied. These measurements were compared with a quantitative model describing both the performance of the ICS sensor and the effects of sample flow rate. It was found that the sample flow rate was a major determinant in the performance of the sensor. Good agreement was obtained with the experimental results by using streptavidin "on rates" of 107M-1s-1, and "off rates" of less than 105s-1 for the streptavidin attachment to the biotinylated membrane capture sites. Two dimensional "on rates" of 10-10cm2s-1 and "off rates" of less than 105s-1 were used to simulate cross linking of the ion channels to the capture sites on the membrane. For the present flow cell geometry, flow rates in the range 40l/min to 900l/min were required to achieve optimal detection sensitivity depending on the streptavidin concentration and capture site densities. The required minimum flow rate becomes greater as the capture site density is increased and the streptavidin concentration is decreased. These results show the potential for an ICS to achieve sensitivities of 100fM with practical sample volumes of 10ul to 100ul provided the sensor size is reduced to the scale of 0.1mm.Cornell B, Braach-Maksvytis V, King L, Osman P, Raguse B, Wieczorek L, and Pace R. "A biosensor that uses ion-channel switches" Nature 1997, 387, 580.
Poster 15Magnotech™: reliable and fast magnetic point-of-care biosensor technology. Evers TH1, Bruls D1, Neijzen J1, Ovsyanko M1, Craus B1, Nieuwenhuis JH2, Dekkers DWC3, Hefti MH3, Martens M3. 1Philips Research, Eindhoven, Netherlands, 2Philips Healthcare Incubator, Eindhoven, Netherlands, and 3Future Diagnostics BV, Wijchen, Netherlands.
Point-of-care in vitro diagnostics applications are highly demanding, requiring tests that are both fast and highly sensitive. Aimed specifically at these applications, Philips Research has developed a novel magnetic biosensor technology that enables high-quality test results near the patient within minutes.The Magnotech technology is based on the controlled movement of magnetic particles in a stationary fluid sample. The large surface area of antibody-functionalized super-paramagnetic nanoparticles is exploited for an efficient capture of target molecules. Electromagnets then rapidly pull the particles towards the sensor surface, where second antibodies capture the particles with a bound target molecule. After controlled surface interaction, unbound and non-specifically bound particles are removed from the surface by a magnetic stringency step. The remaining specifically bound particles are detected on the surface by evanescent-field optical imaging with real-time readout.In this presentation we will describe the integrated biosensor technology and highlight solutions for two types of assay multiplexing, namely chamber multiplexing as well as spot multiplexing. Chamber multiplexing refers to the splitting of the fluid sample over separate one-microliter reaction chambers, which enables the implementation of four completely independent assays in a cartridge from a single drop of sample fluid. Spot multiplexing refers to the readout of up to 30 antibody spots per microliter chamber. The double-multiplexing technology opens up a large application space and offers the flexibility to have integrated controls. We will describe how all steps of the assay are accurately steered by magnetic forces and how the cartridge temperature is accurately controlled. The cost-effective plastic cartridge contains all reagents in a dry form and is filled by capillary forces, requiring a total sample volume of only a few microliters.With the combination of actuated magnetic particles and simple optical readout in a plastic cartridge, we show 5 minute detection of picomolar concentrations of cardiac Troponin I in pure plasma. Because of the speed, ease-of-use, multiplexing capability, and high analytical sensitivity of the method, the technology is well suited for demanding point-of-care medical diagnostic applications which require a short turnaround time, high performance and reliability.
Poster 16A novel point-of-care assay measurement device using imaging technology. Dylewski, S, Alverix, Inc., San Jose, CA.
Lateral flow assays are well established and have enabled a wide variety of point-of-care tests. Despite the benefits of faster results, adoption can be limited by:• Complicated test procedures or sample preparation• Lack of automatic record keeping• Insufficient sensitivity• Difficult interpretation of quantized line strengths• Lack of sophisticated measurement tools for assay developmentAlverix has developed a low-cost, flexible platform for assay measurement which helps to solve some of these problems. The system uses an image sensor backed by a microcontroller to quantify and report the assay line strengths. When using this assay measurement platform, there are many advantages, including automatic record keeping and quality control, increased sensitivity, and accurate quantization. Both reflective and fluorescent instruments are being made today. Without the ability to accurately measure assay performance, scientists spend excessive time developing assays, thus reducing their market competitiveness. The Alverix platform can be used during assay development to generate real-time quantized performance results and quickly optimize a wide variety of lateral flow assays. With this integrated platform, algorithms are designed or modified quickly during assay development, and then encoded into a low-cost handheld instrument for commercial use. This poster will show results from both reflective and fluorescent assays, quantized measurements, linearity over a high dynamic range, and real-time analysis of lateral flow assays. These techniques will enable assay developers to achieve a faster and better understanding of their assay performance.
Poster 17Rapid fabrication of protein arrays for multiplexed assays. Nath N, Creswell D, Simpson D. Urh M. Promega Corporation, Madison, WI.
Objective: The goal of this study is to describe use of HaloTag™ technology for rapid creation of protein arrays by integrating protein expression and capture steps. No prior protein purification steps are required and proteins are captured in a covalent and oriented fashion.Clinical Relevance: Protein arrays consist of thousands of proteins immobilized on a surface that are probed for proteome-scale detection of protein-interaction networks. Researchers are increasingly using these arrays in clinical diagnostic applications by profiling for antibody response to several cancers antigens, autoimmune disorder and infectious agents. Protein arrays have the advantage of miniaturization and multiplexing, enabling large amounts of information to be generated rapidly and from small amounts of clinical sample. Method: The HaloTag protein is a 33 kDa engineered derivative of bacterial hydrolase that forms a covalent bond with its ligand. In the current study we expressed five different proteins fused with HaloTag protein in E.coli or in cell-free protein expression systems. Two of the five proteins were model proteins - GST and Protein G. The other three proteins were: S100A7 a breast cancer marker; p53 a generic cancer marker; and EBNA a marker for Epstein-Barr virus (EBV) associated malignancies. To create protein arrays, lysates containing HaloTag fusion proteins are simple incubated for 1.0hr on a glass slide coated with HaloTag ligand activated hydrogel. HaloTag fusion proteins bind specifically and covalently to the ligand while the hydrogel coating minimizes the non-specific binding. Results: All five proteins fused to HaloTag could be expressed and directly captured either from cell free expression systems or lysed E.coli cultures even at low concentration (<10µg/ml). n="7)." n="12)" n="9)." n="4).">
Poster 23Quantitative detection of PCA3, PSA, and internal control in a quantitative, multiplex, universal real-time transcription-meditated amplification assay format. Nelson NC, Lyakhov DL, Phelps SS, Chelliserrykattil J, Carlson JD, Kaminsky MB, Gordon PC, Hashima SM, Ngo TV, Brentano ST. Gen-Probe Incorporated, San Diego, CA.
PCA3 is a very sensitive and specific marker that aids in the detection of prostate tumors1. In men undergoing repeat prostate biopsy, a recent study has shown that the PCA3 score (ratio of PCA3 to PSA (prostate-specific antigen) present in whole urine after a digital rectal exam) is superior for predicting biopsy outcome when compared to serum PSA determination2. It was the objective of the work presented here to determine if the universal real-time TMA format3 could be used to simultaneously quantitate PCA3 and PSA in the same reaction vessel.Two different modes of the universal real-time TMA format were tested in proof of principle studies. In mode 1, the non-T7 primers and T7 providers for all analytes contained universal sequence tags. In mode 2, only the non-T7 primers contained tags. Primers/providers containing tags were bound to their respective targets (in vitro transcripts used in these studies) during target capture, and amplification reactions were performed using a single universal NT7/T7 pair (mode 1) or a single universal NT7 primer and 3 specific T7 providers (mode 2). In all assays, an internal control was present at 1-4 x 104 copies/reaction. PCA3 and PSA were present at between 102 and 106 copies/reaction, mixed in different proportions. Different-colored fluorescent labels were used to detect each analyte.PCA3, PSA, and internal control (N = 8) were all successfully amplified and quantitatively detected across a 4-log dynamic range. Accuracy (± 0.25 log copy) and precision (standard deviation ≤ 0.25 log copy number) were within desired limits for this feasibility study. The observed interference between PCA3 and PSA was very low, even when the analytes were present at 1000-fold different levels.In conclusion, the feasibility of utilizing a universal real-time TMA format for the quantitative detection of PCA3, PSA, and an internal control in the same reaction vessel has been demonstrated. This assay, currently in research only, may show great utility for use in prostate cancer diagnosis.1J.B. de Kok et al., DD3 (PCA3), a very sensitive and specific marker to detect prostate tumors, Cancer Res 62 (2002), p. 26952L.S. Marks et al., PCA3 molecular urine assay for prostate cancer in men undergoing repeat biopsy, Urology 69 (2007), p. 532.3Lyakhov, D, et al., Development of a quantitative, universal real time Transcription-Mediated Amplification (TMA)-based format for use in multiplex nucleic acid assays, Oak Ridge Conference poster (2009).
Poster 24Performance evaluation of three LC-MS methods implemented on Ion Trap Mass Spectrometer for drug testing in urine. Jiang G, Kozak M, Nimkar S. Thermo Fisher Scientific, San José, CA.
Novel Aspect: Evaluation of effectiveness of MS2, MS3 and MS2/MS3 modes using ion trap mass spectrometer for unknown compound identification in screening applications.Introduction and Objective: Drug identification in complex samples is one of most important applications in toxicology laboratories. Three different methods identifying compounds base on MS2, MS3 and MS2/MS3 spectra and retention time were evaluated for drug testing in urine samples.Method: Three methods collecting MS2, MS3 and MS2/MS3 spectra for compounds identification were developed on LXQ Ion Trap mass spectrometer. All methods included a scan depended experiment in polarity switch mode and a 13 minutes LC gradient using 50x2.1mm PFP column. Spectra were collected in compound specific retention time windows with compound optimized collision energy.Validation: Urine samples were spiked with randomly selected sets of compounds to specified concentrations, processed with SPE procedure and analyzed. Compounds identification was done automatically by a software that compared collected spectra and retention time with the spectra in the library spectra. Performance of the method was validated based on number of compounds detected in the known samples.Results and Conclusion: All 300 compounds in a sample could be easily identified with a method that utilized MS2 and RT. Method based on MS3 and MS2/MS3 spectra could identify 80-100 compounds. The low threshold for data collection set up to achieve better LOD, led to identification of some endogenous compounds in the sample matrix The lower duty cycle of MS2/MS3 method decreased the number of spectra collected for target compounds resulting in lower hit rate. Methods implementing MS3 or MS2/MS3 spectra for compounds identification are less affected by interferences producing combined spectra than method identifying compounds base on MS2 spectra. The identification rate of MS3 or MS2/MS3 method can be increased by raising LOD.
Poster 25A novel, miniaturized, potentiometric, sensor for immunoassays. Piran U, Frew E, Rehak M, Morris R, Gover A. DxTech LLC, Merrimack, NH.
DxTech is developing a cartridge based Point of Care (POC) system for immunoassays and routine clinical chemistries in the physician's office. The system can perform multiple assays in less than 10 minutes using only a small volume of blood on one common biosensor platform. The core of the system is a potentiometric, electrochemical sensor which provides high sensitivity and broad dynamic range. The screen printed sensor, about 2mm in diameter, consists of a polypyrrole layer coated onto a silver electrode. For immunoassay applications antibodies are coated onto the polypyrrole surface to permit capture of the appropriate target molecule. A generic enzyme immunoassay reaction system is then used that consists of antibody conjugated horse radish peroxidase with hydrogen peroxide and traditional organic substrates that generate up to 200mV signal at the sensor.Each sensor is incorporated into a flow cell of 7ul volume on the cartridge. The injection molded cartridge, which supports multiple assays, contains a filter for plasma separation, blister packs to store reagents, fluidic reservoirs, channels and valves to direct plasma/reagent to the sensor array and waste areas for safe onboard storage of liquids. After a whole blood sample is loaded on the cartridge, and the cartridge placed into the reader, all subsequent activities used to generate a result are automatic. The reader maintains the cartridge at 37C and contains all the electrical and mechanical components to externally actuate the cartridge. Filtered plasma and reagents are successively flowed across the sensors within a flow cell to generate a result. Continuous flow ensures that the surface of the sensor is constantly refreshed with plasma or reagents to maximize the rate of the reactions and, combined with the high sensitivity sensor, produces a result in nine minutes. Up to three sensors per assay are currently incorporated into each flow cell. One sensor is dedicated to target capture while the remaining sensors may be used for either calibration or to provide high and low sensitivity sensors for TSH.DxTech is currently developing a TSH assay for a thyroid panel. Preliminary data was collected using a flow cell breadboard as well as an integrated cartridge. Flow cell breadboard data (Table 1) yield almost third generation performance with plasma standards in seven minutes with a single sensor. The cartridge data (Table 2) yield second generation performance with TSH in Hepes buffer in seven minutes using a combination of a high sensitivity (50ug/ml anti-TSH sensor surface antibody) and low sensitivity sensor (1ug/ml anti-TSH sensor surface antibody).
Poster 26Evaluation of Nanogen's NexusDx® multianalyte POC assay system. Egan R1, Vukajlovich S1, Shaw J1, Madsen R1, Krodel E2, Belenky A1, Bluestein B1, Lidgard G1. 1Nanogen, Inc., San Diego, CA, and 2HX Diagnostics, Emeryville, CA.
Nanogen, working in collaboration with our partners, has developed a 3rd generation POC assay format that better meets the needs of patients and clinicians when compared to existing POC assays. The new NexusDx technology and format has unique features that, taken in combination, result in an important evolutionary move for POC testing with significantly improved performance. For example in a demonstration of sensitivity, the new assay system detected <1x10-18>99.5% and an analytical sensitivity that ranges between 2 and 3 logs higher than currently marketed assays. The assay simultaneously tests for influenza type A and type B, and the subtypes of A, H1 and H3. A sensitive set of H5 reagents has also been developed. The following table shows the performance of the type A and type B reagents.
fluID Rapid Influenza Test Vs a marketed POC Influenza assay and PCR:Virus Strain (Titer in TCID50 units) B/Ohio . Titer Diff. vs fluID , A/Wisconsin . Titer Diff. . vs fluID A/New Caledonia . Titer Diff. .vs fluIDTiter in POC Rapid Test s 40,000 +>1,000X 40,000 +>100X 40,000 + 100XTiter in FluID Assay 6.0 200 400 Titer in PCR Assay 0.15 -40X 8 -25X 4 - 100X
NT-proBNP and Troponin I: The NexusDx system was designed as a quantitative platform and yields performance similar to full scale analytical systems in a low cost rapid POC assay system. Data developed by Nanogen using early development lots has shown that the NexusDx system can detect NT-proBNP at a limit of quantitation of 3.5 pg/mL, a limit of detection of 0.3 pg/mL, 3.5 log range, and have CV's of less than 5%. For TnI the limit of quantitation was determined to be 30 pg/mL. In summary: The NexusDx multianalyte POC assay system developed by Nanogen, in association with our partners, is a unique platform that builds upon well established lateral flow technology in a way that yields large system performance in a rapid simple to use POC system for a wide array of analytes (proteins, antibodies and nucleic acid.Developed in collaboration with HX Diagnostics, Inc. and funded in part by the Biomedical Advanced Research and Development Authority Office (BARDA) of Assistant Secretary for Preparedness and Response (ASPR), U.S. Department of Health and Human Services (HHS), and the Centers for Disease control (CDC) for advanced product development under contract No. HHS200-2007-19345. BARDA and the CDC are not responsible for the content in this Abstract. NexusDx® is a registered trademark of Nanogen, Inc. fluIDTM is a trademark of HX Diagnostics, Inc.The clinical utility for these assays has not been determined.
Poster 27Universal nanoparticle-based platform for rapid clinical diagnostic. Lowery TJ, Kumar S, Mozeleski B, Taktak S, Fritzemeier M, Rittershaus C, Demas V, Prado PJ. T2 Biosystems, Cambridge, MA.
T2 Biosystems' universal NanoDx detection platform consists of a portable Magnetic Resonance (MR) device that analyzes nanoparticle-based assays. The proprietary assays contain superparamagnetic nanoparticles decorated with selective binding agents that sensitize the particles to the desired target analyte. In the presence of the analyte, the particles undergo a transition in their clustering state, which can be detected by measuring a change in the MR signal from surrounding water molecules. This technology can be used, e.g., to detect small molecules, proteins, nucleic acids, and ions from a single sample.
T2 Biosystems' diagnosis is fully automated. The operator loads the specimen, such as whole blood or urine, onto the cartridge and inserts the cartridge into the instrument. After a short period of time, the instrument provides a quantified result available at the point of care or reported through the laboratory information system. These devices are ideal for use in doctor's offices, emergency clinics, and ambulances.
In this presentation, we introduce the NanoDx platform and describe the underlying technology. We will present data for a menu of nanoparticle assays that detect proteins and small molecules with nanomolar to picomolar sensitivity in whole blood and urine. Additionally, we will introduce our blood coagulation time and waveform measurements and a preliminary correlation study.
Poster 28Spinning disc platform for digital PCR. Sundberg SO1, Gale BK2, Wittwer CT3. 1Bioengineering, 2Mechanical Engineering, 3Pathology, University of Utah, Salt Lake City, UT.
Motivation: Digital PCR is capable of detecting single DNA molecules. Rare mutations within an excess of normal DNA can be detected and genetic allelic imbalance can be quantified. This process is expensive and difficult because of the thousands of reactions necessary. Although dilutions can be used to achieve single DNA copy reactions, reduction in sample volume is another solution. The spinning disc platform uses an inexpensive rotating disc to partition the sample into a thousand or more micro-sized wells. Materials and Methods: A knife plotter patterned a PETG sheet (125 microns thick) with a spiraling channel having 1000 wells (30 nl/well), facing radially outward and tangential along the spiral. This patterned sheet was then thermally bonded between two similar PETG sheets by lamination, thus creating the rotating disc. PCR solution with the dye LCGreen® Plus was pipetted into an inlet port towards the center of the disc and then spun at 3000 rpm to load each well. A modified air thermal cycler was used for PCR amplification (40 cycles in 16 minutes) and the disc was interrogated using a CCD camera image to determine how many wells fluoresce for quantification. Results and Conclusions: All wells were filled with a volume CV of 20%. The Figure shows single DNA molecule detection is possible with target dilution down to less than an average of 1 copy/well. The spinning disc platform is capable of partitioning and quantifying a sample and can be applied to multiple digital PCR applications. The spinning disc platform is an improvement over other volume limiting platforms because no valving or pumping is required. Furthermore, rapid air cycle PCR is possible for increased speed and throughput.
Novel AspectEvaluation and comparison of three sample preparation methods implemented in screening application on ion tap mass spectrometer Introduction and ObjectiveLC-MS is a powerful technique for drug screening. New, sensitive MS/MS systems enable detection of drugs at low levels. However the quality of LC-MS data collected in screening application is largely affected by sample preparation method. Three different urine sample preparation methods implemented in toxicology screening were evaluated on set of 300 basic, neutral and acidic compounds. MethodWe compared 3 sample preparation methods to find the most robust, easy to use and efficient method to acquire quality MS/MS data. Solid phase extraction, liquid-liquid extraction and on line extraction methods were compared. SPE method for basic, neutral and acidic compounds implementing cation exchange, non-polar cartages was developed in house. Commercially available ToxiTube product was used to evaluate liquid-liquid extraction. TurboFlow method on Aria TLX system using dual columns was developed in house.ValidationUrine samples spiked to concentrations 1-100 ng/mL with 10 randomly selected compounds were processed with SPE, LLE and on line extraction. Processed samples were then analyzed with LC-MS screening method implemented on Quantum LXQ mass spectrometer. Compounds were identified base on MS2 spectra and retention time. In addition diluted urine samples were analyzed for data comparison.Results and ConclusionAll evaluated sample preparation methods allowed for identification limit required in toxicology screening. SPE requires longest time but it can be automated for better efficacy. ToxiTube method gives similar results to SPE but it is more efficient. On line extraction is the most efficient and most cost effective method. The on line methods can also be multiplexed to double or quadrupole sample throughput.
Poster 31LAMP without electric heat: a chemically-heated, non-instrumented nucleic-acid amplification assay platform for point of care use. LaBarre PD,1 Gerlach J,1 Wilmoth J,1 Beddoe A, 1 Singleton JL,1 Weigl BH1. 1PATH, Seattle, WA.
Many infectious diseases that impact global health are best diagnosed through pathogen-specific nucleic-acid (NA) detection. However, highly accurate, disposable nucleic-acid amplification tests (NAATs) are usually not accessible to populations in low-resource settings. By reaching this "first completely non-instrumented NAAT" milestone, we have verified the function of the critical component of a diagnostic platform that can utilized for a wide selection of NAATs. This platform will be as simple to use as a strip test, and free from the traditional power requirements that constrain conventional NAATS to well-equipped laboratories. We envision a low-cost NAAT platform that can be stockpiled indefinitely and used at the point of care (POC) with minimal training. We have achieved the first complete, non-instrumented NAAT using a calcium oxide heat source thermally linked to a proprietary phase change material (PCM). These two components alone maintain a thermal profile suitable for the loop mediated isothermal amplification (LAMP) assay. Starting with computational fluid dynamics (CFD) analysis, we identified nominal geometry for the exothermic reaction chamber, PCM chamber, thermal insulation and packaging. Using this model, we designed and fabricated an alpha prototype assay platform. We have verified the function of this multi-pathogen-capable platform with both fluorescent and visual turbidity indications using samples spiked with malaria DNA. Both the exothermically heated platform samples and samples heated on a Perkin-Elmer GeneAmp9600 thermocycler were first incubated at 62°C for 45 minutes, then heated to 95°C to terminate enzyme activity, then analyzed. Results from the exothermically heated, non-instrumented platform were comparable to those from the thermocycler. Furthermore, we have identified and tested appropriate combinations of exothermic materials and PCMs which can be used to establish non-instrumented thermal profiles appropriate for denaturization and other isothermal diagnostic processes. These developments will enable POC diagnostics using accurate NAATs which, until now have required a well equipped laboratory. The aim of this system is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where assays such as immunochromatographic strip tests are successfully used, but where there is no access to the infrastructure and logistics required to operate and maintain instrument-based diagnostics. Minimally trained health workers will be able to operate this low cost, highly accurate, disposable test with no additional instrumentation. As LAMP has been demonstrated to be an accurate NAAT for diagnosis of over 20 infectious diseases, the platform we have successfully tested is not limited to detection of one specific pathogen. While we verified the function of this technology using malaria, other pathogens such as HIV, MRSA, and TB can be detected as well since the thermal profile required for detection using LAMP is independent of the pathogen. We envision several commercial configurations of this technology including a multi-pathogen-capable, multi-sample, exothermically heated incubator and a self-contained, pathogen specific, disposable NAAT with dried-down reagents.
Poster 32Bacteria detection with integrated on-chip sample preparation, PCR, and fluorescence detection with novel remote valve switching fluidic control. Sauer-Budge AF1,2, Klapperich CM2,3, Mirer PL1, Chatterjee A3, Sharon A1,3. 1Fraunhofer Center for Manufacturing Innovation, Brookline, MA, 2Biomedical Engineering Department, Boston University, Boston, MA, and 3Mechanical Engineering Department, Boston University, Boston, MA.
Currently, the majority of the bacterial infections diagnoses are conducted via culture which takes many hours to days to identify the bacteria. Thus, physicians will typically prescribe an initial broad-spectrum antibiotic drug therapy at the initial examination and then change the therapy as needed upon receipt of the culture results. This practice contributes to the rise in antibiotic resistance and is often ineffective at treating the patient. To address these concerns, rapid diagnostics have been developed; both immunoassays and nucleic acid based tests. The immunoassays often suffer from inadequate sensitivity and/or specificity, while the nucleic acid based tests (NAT) are expensive. The NATs require specialized lab space, expensive reagents, and extensively trained technicians to appropriately conduct the assays. In practice, these factors lead to batch processing once a day in most clinical labs making the effective turn-around-time 24 hours. To address these challenges and provide a truly rapid test that meets the demands of high sensitivity and specificity, many have proposed to develop a point-of-care molecular diagnostic that automates the sample preparation, nucleic acid amplification, and detection in a low cost miniaturized lab-on-a-chip format. The benefits of this format include reduced costs (less technician time, no need for specialized facilities, less reagent use), improved reproducibility, and fast results. However, much of the work in this field has not yet resulted in a completely integrated lab-on-a-chip or a design that is truly low cost to manufacture. We have developed a completely integrated lab-on-a-chip and associated instrument for the detection of bacteria from liquid samples. The system conducts bacterial lysis, nucleic acid isolation and concentration, polymerase chain reaction (PCR), and end-point fluorescent detection of the target amplicon. To enable truly low-cost manufacture of the single-use disposable chip, we designed the plastic chip in a planar format without any complicated active components to be amenable to injection molding and utilized a novel porous polymer monolith (PPM) embedded with silica that has been shown to lyse bacteria and isolate the nucleic acids from clinical samples (Kulinski et al. Biomed Microdevices epub 1/9/09). The chip is made in Zeonex, which is a thermoplastic with a high melting temperature to allow PCR, a good UV transmissibility for UV-curing of the PPM, and low auto-fluorescence to allow fluorescence detection of the amplicon. We built a prototype instrument to automate the control of the fluids, temperature cycling, and optical detection with the capability of accommodating various chips designs. We employ a novel method that enables fluid control without valves or pumps on the chip. The PCR thermal cycling was achieved with a ceramic heater and air cooling, while end-point fluorescence detection was accomplished with an optical spectrometer. The integrated functionality of the chip was demonstrated using Bacillus subtilis as a model bacterial target. A Taqman assay was employed on-chip to detect the liberated bacterial DNA.
Poster 33 Rapid concentration of bacteria in a disposable microfluidic device using enhanced evaporation technique for a Point-of-Care (POC) infectious disease diagnostic device.Do J, Zhang JY, Klapperich CM. Department of Biomedical Engineering, Boston University, Boston, MA.
We report a low cost, disposable polymer microfluidic sample preparation device to perform rapid concentration of bacteria from biological samples using enhanced evaporation.Conventional diagnosis of infectious diseases involves extraction, incubated culture and growth, and morphological, microscopic or biochemical identification of the potential pathogen from patients' bodily samples. These conventional approaches make diagnosis of infectious diseases risky, slow, costly, and pose a great challenge to detecting infectious diseases at an early. Therefore, there is an urgent need for a POC infectious disease diagnostic device with a sample preparation component that replaces incubated culture. Concentration is easily accomplished in a laboratory setting, with equipment such as the centrifuge. However, a centrifuge is not portable, prone to contamination, and labor intensive at the POC. By contrast, microfluidic sample concentration is a portable and automatable processThe developed device includes a liquid sample layer and an airflow layer, between which a hydrophobic porous membrane layer is sandwiched. The convective airflow enhances evaporation of water across the liquid/gas interface at the membrane. Concurrently, liquid flows from inlet to outlet to compensate for the evaporation. In the process, the hydrodynamic force drags the analyte along with the liquid meniscus.The device was tested using E. coli. Concentration efficiency was defined as the ratio of bacteria concentrated into a volume of 500 nl divided by the initial number of bacteria in 100 µl. Serial dilutions of fluorescently tagged bacteria were made, run through the chip and imaged with fluorescent microscopy. Figure 1a shows fluorescent images of the outlet and along the channel after enrichment. Figure 1b shows the percentage recovery efficiency versus initial bacteria concentration. The recovery efficiency is above 90% for initial concentrations lower than 6.3 x 104 cfu/ml. 100 µl initial volumes of bacteria solution at 100 cfu/ml were concentrated into 500 nL droplets with greater than 90% efficiency in 15 min.
A fast and reliable analysis to formulate the right diagnosis or to decide the correct therapy can be crucial for the patients' survival in many cases. For example the timeliness in a correct diagnosis can be essential in the discrimination of viral and bacterial sepsis in intensive care patients. In this case, the simultaneous measurements and quantification of analytes such as C-reactive protein (CRP), procalcitonin (PCT), TNFα, myeloperoxidase, interleukines and neopterin can be extremely important for physicians to define the etiology of severe infection.Among the above mentioned biomarkers, CRP and PCT are the parameters analysed in this paper. The circulating concentration of human CRP and PCT are 1-10 mg L-1 and 0.1 μg L-1, respectively. They may rise from normal levels up to about 500 mg L-1 and 100 μg L-1, respectively. The optical platform developed for the measurement of these two parameters is constituted by a two-piece of polymethylmethacrylate (PMMA) chip, with microchannels through which the analysed sample flows. The sensing layer, where the immuno-chemical reaction takes place, is located on the bottom side of the upper piece of the PMMA chip. The excitation is made with a 635-nm laser-diode radiation that goes perpendicular to the sensing layer. The emitted fluorescence, which comes from the sensing layer when the specific biologic interaction takes place, is mainly coupled to the PMMA cover of the chip. This occurs thanks to the fluorescence anisotropy, which is exhibited whenever the fluorophore is located at a distance of a medium interface of the order of the wavelength. The fluorescent signal is laterally collected by a single plastic optical fibre connected to a Hamamatsu optical spectrum analyser. In figure 1 an example of the calibration curve obtained for CRP is shown. A limit of quantification of 13 µg L-1 in a working range of 0.01-500 mg L-1was achieved.
Poster 35Instability of human plasma peptide biomarkers caused by intrinsic peptidase activity. Yi J, Craft D, O'Mullan P, Ju G, Liu ZX. BD Diagnostics, Franklin Lakes, NJ.
Peptide biomarkers, like other endogenous peptides in human plasma, are subjected to degradation caused by intrinsic proteolysis. In this study, we directly investigated the stability of peptide biomarkers by spiking a peptide into human plasma and serum samples and then monitoring its time-dependent change. Peptide biomarkers including FPA, C3, C4, BNP, GLP-1, ect, in a conventional serum and plasma samples either with citrate, heparin, or EDTA as the anticoagulant, or EDTA plus protease inhibitors (inhibited plasma), were investigated. The parent peptides and their degradation products were measured and monitored using time-course MALDI-TOF MS analysis. Kinetics analysis indicates that intrinsic peptidases cause an observed first-order Sequential Multiple-Step Reaction (SMSR). Modeling analysis of the SMSR demonstrates that step reactions differ in their kinetic rate constants, suggesting a significant contribution of the truncated end residue on the substrate specificity of the intrinsic peptidase(s). While the degradation of peptide biomarkers is observed in a broad spectrum in traditional blood samples, the stabilization of the peptide biomarkers is a key component for biomarker development. This work demonstrates the inherent variability of peptide biomarkers in plasma and serum and the use of protease inhibitors to minimize this variability.
Keywords: peptide biomarker, stability, time-course MS, serum and plasma, sequential multi-step reaction, or SMSR
Poster 36ReLIA: a portable low cost point of care immunoassay platform with high sensitivity, low CV, broad dynamic range and connectivity. Liu H, Sierra GH, Ye J, Zhang JZ, Rutter WJ. ReLIA Diagnostic Systems, Inc., Burlingame, CA.
Low sensitivity, high CV, narrow dynamic range and no connectivity are the limitations of current point-of-care (POC) systems. These characteristics have hindered the adoption by clinical laboratories. ReLIA has developed a low cost high performance portable immunoassay system for POC that provides the sensitivity, precision, accuracy and reliability of expensive and complex central laboratory systems, with the advantage of on site testing by non-technical personnel (designed to be CLIA waived). ReLIA III employs a dual laser-induced fluorescence technology to dramatically increase the signal to noise ratio, considerably reduce CV, and expand the dynamic range up to 3 logs without having to dilute samples. With such an advanced lateral flow design, it allows quantitative point-of-care diagnostics of whole blood samples in less than 10 minutes, and more accurate and sensitive analysis of analytes. The system is expected to reach an analytical sensitivity of at least 10 pg/ml with CV less than 5%. The ReLIA system is capable of testing up to 10 disease markers simultaneously. The system satisfies the increasing regulatory and customer requirements for quality control, result verification and system validation. It automatically carries out up to 40 different internal control calibrations. No standard curves or other control measurements are required to be carried out by users. Assay specific information is stored in the instrument via a bar-code on each cassette. The instrument detects the addition of samples, times the assays and then automatically reads the cassettes. The system is very robust, and capable of being remotely monitored and/or controlled by the ReLIA Call Center. Consequently, the system does not require on-site services. Internet connectivity is standard, and wireless connection is optional. The original validated data can be transmitted to any desired location for further analysis, or integration into large data sets for disease management or control. In addition, ReLIA system can be coupled with iMetrikus networks for healthcare IT management in a corporate geographical, countrywide or worldwide basis. High quality results, improved convenience, superior data management, and cost effectiveness characterize the ReLIA system.
Poster 37Disposable bacterial detection device applying DNA extraction and isothermal helicase-dependent amplification. Mahalanabis M1, Muayad H1, Klapperich CM1,2. 1Departments of Biomedical Engineering, and 2Manufacturing Engineering, Boston University, Boston, MA
Point-of-care microfluidic devices for the detection of microbial pathogens of humans are needed as a practical alternative to current large bench-scale techniques. We demonstrate proof-of-concept of a complete microfluidic disposable detection system using Escherichia coli (E. coli) as a representative bacterial pathogen. The microfluidic device is a cyclic olefin polymer chip containing features generated from hot embossing with an epoxy resin mold patterned from PDMS and soft lithography. Cell lysis and DNA extraction are coupled on silica polymer monolith columns using a combination of mechanical and chemical lysis and solid-phase extraction of DNA. The DNA is then directly eluted into a 65C heated chamber for helicase-dependent amplification (HDA) which is detected by fluorescence imaging or agarose gel electrophoresis off-chip. Three different HDA primer sets were designed for the single copy chromosomal gene, dxs, and DNA was isolated from 105 colony forming units (CFU)/mL of E. coli liquid culture. The chip extraction was comparable to a commercial bacterial DNA isolation kit with the DNA concentration at 10ng/uL. HDA on-chip with 10uL of the eluted DNA (100ng) generated the expected 100 base pair size products from all three primer designs. The chip results were identical to results from HDA reactions run off-chip in a standard PCR thermocycler. Due to the single incubation temperature of HDA, it is a powerful diagnostic tool that is more amenable to a simple microfluidic platform than other common amplification methods like PCR that require cycling between multiple temperatures. This work demonstrates the use of a fully integrated microfluidic device to detect human pathogens of interest in a module that combines cell lysis, DNA extraction, and amplification.
Poster 38The evaluation of platelet function on two diagnostic platforms – VerifyNow® V's TEG®.Carville DGM1, Walker CT2. 1Indiana University South Bend, South Bend, IN, and 2Heart First, Baptist Hospital, Pensacola, FL.
Introduction: Due to the clinical significance of platelet function testing for cardiac surgical procedures including cardiopulmonary bypass (CPB) and percutaneous coronary intervention (PCI) companies have invested serious research in developing platforms to evaluate plate function. Such platforms include light transmission aggregometry (LTA) and variations thereof (Plateletworks, Helena POC, Beaumont, TX); the Dade PFA 100 (Dade-Behring, IL); HMS (Medtronic, Minneapolis, MS); new comer Thromboguide (Thrombovision, Houston, TX); and also VerifyNow (Accumetrics, San Diego, CA) and the Thromboelastograph (TEG) (Haemoscope, Niles, IL). In this study we evaluated patients undergoing CPB using both the VerifyNow system and the TEG to determine if there was a difference in the platelet function response.
Methods: Patients who were scheduled to undergo CPB with the potential of drug eluting stent (DES) placement were recruited following informed consent at Baptist Hospital, Pensacola, FL. Patients demographics were: age 30-76; had a history of coronary artery disease and; were administered clopidogrel. Other complications included diabetes, hypertension, obesity and stent placement. Patients were evaluated using the VerifyNow P2Y12 cartridge (Accumetrics, San Diego, CA) and TEG platelet mapping (Haemoscope, Niles, IL) and tested for % platelet inhibition. Results as mean ± standard deviation are shown below.
Results: Platelet % inhibition was observed at 19.7 (±16.7) for the VerifyNow platform and 23.2 (±8.8) for TEG. However, in regression analysis the r value was reported at 0.041 suggesting that there is negligible correlation between the platforms, which is most likely a reflection of the semi-quantitative nature of the VerifyNow system.
Conclusions: These data demonstrate that the use of various platforms (especially at the point-of-care) need to be carefully evaluated by the institution and compared with the gold-standard of platelet function testing LTA. Albeit this was a small study caution is advised when determining appropriate therapies in these clinical environments.
Discussion: Due to the recent understanding of platelet therapy resistance (between 5-60%) platelet function is clinically required in settings where patients receive anti-platelet therapy including the thyroperidines, the GPIIb/IIIa and aspirin among others. Institutions need to follow appropriate procedures and establish their own guidelines for treatment. However, what is clinically important are outcome studies at six and twelve months subsequent to the procedure to determine how well patients on anti-platelet therapy are served and what is the optimal platform for triage decisions.
Poster 39Evaluating coagulopathies and anticoagulation (UFH & LMWH) therapy using a novel point-of-care (POC) intrinsic and extrinsic coagulation analyzer. Spencer J1, Ridgway HR1, Rullman RL1, Carville DGM1,2. 1Helena POC, Beaumont, TX, and 2Indiana University South Bend, South Bend, IN.
Thrombosis (adverse coagulation) remains the leading cause of morbidity and mortality in developed countries and is a significant side effect of medical procedures including bypass surgery and trauma. In conjunction with the introduction of novel anticoagulant therapies including low molecular weight heparins (LMWH) and the recombinant hirudins, optimized platforms are required to permit the best possible clinical intervention and improve patient outcomes. Here we describe the use of a single multiple analyte platform for coagulation triage - the Helena Cascade® POC analyzer. This system utilizes cards with a reaction chamber containing all test reagents and paramagnetic iron oxide particles (PIOP), which are reconstituted by the sample and move under the influence of oscillating magnetic fields generated by the analyzer. Clot formation impedes the oscillation producing a signal that is interpreted by a predefined algorithm providing a clot time (secs.).
Clinical samples (N=>50) were compared in both citrated whole blood and plasma for aPTT, PT, ACT and LMWH (enoxaparin) evaluation. Excellent correlations were observed between both sample types: aPTT r = 0.99; PT r = 0.99; ACT r = 0.92. In addition, heparin linearity with the Cascade platform was observed to (final concentration) 0.5U/mL for aPTT and 8U/mL for ACT. The LMWH test card demonstrated linearity to 2.0IU/mL. Data for ACT testing compared with a traditional ACT platform are shown in figure.
Poster 40An instrument-free point-of-care CD4 T-cell test using a novel method for rapid and inexpensive cell counting. Zaugg F, McManus-Munoz S, Yu F, Tobias R, Ruiz-Taylor L, Venkataraman S, Kernen P, Wagner P. Zyomyx, Inc., Hayward, CA.
BACKGROUND: We report here a rapid and simple point-of-care test capable of counting CD4+ T-lymphocytes in whole blood, a key parameter in the treatment and monitoring of those living with HIV/AIDS. We have designed this diagnostic to be instrument-free, a critical factor to ensure the greatest and widespread implementation in rural areas of limited resource countries. Despite initial specifications calling for a semi-quantitative CD4 diagnostic (minimum of one cut-off for initiation of therapy), our test has the potential to become truly quantitative in the clinically relevant adult and pediatric range of 0 to 1000 cells/µL. The test is based on Zyomyx's cell stacking rapid test technology that eliminates the need for reporter reagents and signal intensity calibration, thereby allowing a simple visual readout by the naked eye without any further requirements for separate standards and optical readers.METHODS: Whole blood is collected via fingerprick and a specific volume transferred into a reaction compartment to specifically label (i) monocytes with paramagentic anti-CD14 Dynabeads® (Life Technologies, Inc., Carlsbad, CA) and concurrently (ii) CD4 cells with CD4 Zyobeads™ (Zyomyx, Inc., Hayward). CD4 Zyobeads are composed of large, dense particles (approximately 1000 nm mean diameter, density higher than 10 g/ml) coated with anti-CD4. After 5-10 min of incubation, the whole blood sample containing the bead-bound T-cell population is subjected to a separation device containing a closed-end high-precision glass capillary (80 micron I.D.) filled with a high density medium. Within 3-20 min depending on the exact test configuration, bead-bound T-cells migrate through the medium to the bottom of the capillary thereby creating a closely-packed stack having a length proportional to the CD4 count. Monocytes and red blood cells are retained ensuring that neither cell population contributes to the CD4 stack. Figure 1A is an illustration of the separation device including a photograph showing the morphology of the stack at the endpoint of the assay, and Fig. 1B shows the overall test assembly.
RESULTS: Good correlations between CD4 counts with the predicate method of flow cytometry have been demonstrated (r2>0.90) and transfer of CD4 T-cells into the stack is efficient. In addition, the test shows very effective retention of red blood cells (no lysis required) for several hours after the assay has concluded. The sensitivity limit with the current configuration is estimated to be 38 T-cells / µL. Preliminary stacking height versus CD4 count plots show linearity in the 0-600 range which will be further extended in the future. CONCLUSION: Point-of-care diagnostics for CD4 are essential to meet the urgent global need for antiretroviral delivery and optimal care for the >20 million people living with HIV/AIDS in resource poor countries. For effective implementation in these areas, CD4 point-of-care tests must meet a number of demanding specifications to allow for inexpensive ease-of-use, robustness and precision without the requirement of cold chain shipment and storage. The presented CD4 test not only fulfills these criteria, but also eliminates the need for any reader technology and has the potential to be quantitative over a clinically meaningful range. Future work will be dedicated to further device integration, large-scale production, and performance evaluation in resource poor settings with large numbers of HIV+ donor samples. ACKNOWLEDGMENT: We thank Hans-Georg Batz and Steven D. Reid, CD4 Initiative at Imperial College, London, for their generous support of this project.
Poster 41A rapid, sensitive, multiplexed HIV diagnostic for resource limited settings. Cull MG, Roark C, West AB, Beacon Biotechnology LLC, Aurora, CO.
HIV screening in low resource settings as well as the application of new technology are of fundamental importance in developing the next generation of diagnostic tools to track and manage the treatment of HIV. Beacon Biotechnology has developed disruptive core technology that can be used for a rapid, multiplexed, HIV diagnostic in low-resource settings. The device consists of an inexpensive, disposable CMOS light detector combined with extremely bright luciferase-based diagnostic assays.
The device has been used to detect the presence of HIV specific antibodies in serum, plasma, and whole blood. In proof of principle, the test generated a positive result for the presence of antibodies against both p24 and p51 HIV antigens using a 1/320,000 dilution of patient samples.
The device is inexpensive, disposable, and currently derives all its power from a USB port. The device can perform simultaneous detection on duplicates of over 50 different targets.
Unlike lateral flow devices, the detection of multiple targets will generate information on the stage of infection, and indicate co-infections with other infectious agents such as Hepatitis B and C that could impact therapeutic strategies. Additionally, typing and sub-typing HIV strains will generate epidemiological data to track the spread of HIV and allow gathered information to be quickly uploaded into public health databases from remote locations.
Poster 42DNA sequencing using digital microfluidics. Thwar P1, Rouse JL1, Eckhardt AE1, Srinivasan V1, Pamula VK1, Griffin P2, Fair RB3, Pollack MG1. 1Advanced Liquid Logic, Research Triangle Park, NC, 2Stanford University, Palo Alto, CA, and 3Duke University, Durham, NC.
Digital microfluidics, characterized by precise, programmable manipulation of liquid droplets using electrowetting, is being applied in an increasing number of applications. We report here our progress towards an inexpensive and compact DNA sequencer for clinical applications based on digital microfluidics. DNA sequencing is accomplished by miniaturization and automation of the pyrosequencing reaction. The digital microfluidic chip consists of an array of electrodes fabricated on an inexpensive printed circuit board (PCB). The chip is covered by a transparent top plate and filled with oil to create a microfluidic chamber in which droplets are programmably manipulated (dispensed, transported, merged, split) using electrical fields. Droplets containing one of the four nucleotides are repeatedly added to a droplet containing a primed DNA template strand in the presence of a polymerase. Successful incorporation of a nucleotide is detected through an enzymatic cascade as a luminescent signal using a simple photodetector located above the chip. Immobilization of the DNA on magnetic beads enables the sample to be automatically washed between each nucleotide addition. Using Candida albicans as a model system several different amplicons were sequenced using this technique. Read lengths of up to 50 base pairs have been achieved and efforts are underway to significantly increase both read lengths and throughput. Rapid PCR and sample preparation capabilities were previously demonstrated on the same platform enabling eventual integration of all analysis steps on a single low-cost integrated device. Likewise the instrument which consists essentially of an array of electrical switches and a photodetector can be made extremely compact and inexpensive.
Microfluidic polymerase chain reaction (PCR) devices could be powerful diagnostic tools. Very small amounts of starting sample can be used, thermal cycling time is reduced, and the potential for integration with other functional devices is large. We have developed a computational model of a 30-cycle serpentine channel continuous-flow (CF) PCR device. The three dimensional model was constructed from CAD layouts, and comprises three coupled sub-domains: the lower substrate (Zeonex 690R), fluidic channel (buffer solution), and upper substrate (Zeonex 690R). We applied two heat transfer boundary conditions; continuous heat flux condition at the interface between solid and liquid materials, and convective heat transfer between the external surface of the chip and ambient air. Once the thermal model was built, we used the information to look at the temperature histories of individual DNA "particles" in the flow.
Here, we combine a novel Lagrangian thermal approach with a Langmuir kinetic model to account for a duplex-forming reaction from two single strands as well as monitoring and controlling DNA thermal exposure time at three different temperature zones for denaturation, hybridization and extension. Simulation results were used to inform our chip design, and experimental results were in turn used to improve our computational model. Thermal modeling results now allow us to predict temperature distributions on chip (and thus its suitability for PCR) before fabrication, eliminating several rapid prototyping iterations. In addition, the Lagrangian thermal approach and Langmuir kinetic model also gives information about the temporal temperature variations of each "particle" of DNA. Detailed information about the "DNA denaturation-renaturation hysteresis" as it moves through the fluidic channel can also be calculated. These simulations will allow for reaction specific design of disposable thermoplastic PCR devices.
Figure 1. Time-dependent denaturation-renaturation hysteresis of coiled DNA in one cycle of the 30 cycle PCR channel under three different initial concentrations (5×10-3 M, 5×10-4 M and 5×10-5 M) of the dNTPs, enzymes, and primers.
Poster 44Intra-feature and inter-feature multiplexing using diffractive optics technology: more information from less sample. Lin Y1, Bernstein G1, Pak BJ1, Fu Q2, van Eyk J3, Ndao M4, Vasquez-Camargo F4, Goyette S4, Ward BJ4, Hu W1 and Houle JF1. 1Axela Inc., Toronto, ON, 2Deptartment of Medicine, 3Department of Biological Chemistry, and 3Biomedical Engineering at Johns Hopkins Bayview Proteomics Center, Johns Hopkins University, Baltimore, MD, and 4National Reference Centre for Parasitology/McGill University, Montreal, QC.
It is becoming increasingly clear that panels of analytes offer greater promise for enabling diagnostics with improved sensitivity, specificity and ultimately clinical utility. Diffractive optics technology incorporated in the dotLab® System allows not only for the inter-feature multiplexing with an extended dynamic range, but also for intra-feature multiplexing. By sequentially probing captured analytes for interacting proteins and/or subunits as well as with affinity reagents specific for post-translational modifications, one can transform cumbersome assays into simple and very informative assays. We report two specific examples of intra-feature multiplexing assays.
1. Detection of the cardiac Troponin complex in patients' samples:In the heart, cTnI is part of the troponin (Tn) complex comprising troponin I (cTnI), troponin T(cTnT) and troponin C (TnC) which is released into the blood upon cardiac muscle necrosis and cell death. It has been previously demonstrated that a subcomplex of cTnI-cTnC circulates in the blood. Furthermore, cTnI has been shown to specifically and selectively degrade with increasing severity of AMI. Until now, the technology for direct detection of the cTnI primary sequence integrity and the ternary form of circulating cTnI has been lacking. The question remains whether circulating cTnI is degraded or is bound to cTnC and/or TnT and whether these various circulating forms of cTnI correlate to long term patient outcomes. We have developed a sensitive and simple-to-use method to directly characterize the interactions between cTnI, cTnT and cTnC from clinical samples as well as probe the integrity of cTnI.
2. Seroconversion in Strongyloides stercoralis infected individualsInfected individuals are typically asymptomatic. However, when immunocompromised, the infection can rapidly progress to a hyperinfected and disseminated state, with an associated mortality of approximately 80%. We have developed a rapid serological method of determining Strongyloides infection. The method utilizes a recombinant antigen to capture antibodies from 10 uL of serum. Sequential probing of positive patients samples with isotype specific antibodies (IgG1,2,3,4 and IgE) enabled the determination of antibody isotype profiles in a single assay. This assay successfully detected serum Strongyloides antibodies in less than 30 minutes and showed little cross reactivity to sera from healthy individuals or patients with other parasitic infections. Isotype profiles may improve serodiagnosis and provide information on disease status such as chronicity or drug resistance.
In addition, the ability to independently measure individual immunoassays without the need for a common reporter allows detection of analytes over a very wide dynamic range, up to 9 logs, in a single sample. We have demonstrated simultaneous measurement of proteins from micromolar through picomolar levels in a single sample utilizing both intra and inter-feature multiplexing. Preliminary data will be shown. Finally, these capabilities coupled with the simple robust optics technology at the core of the current compact bench-top platform can be readily deployed, as in the current stand-alone system, and has the potential to be incorporated into various other platforms in the central laboratory and near-patient testing.
Poster 45A rapid and compact on-site molecular analyzer. Wong S1, Ugaz V2, Wallek B1, Shandy S1, Ragucci T1, Soirez L1. 1Lynntech, Inc., College Station, TX, and 2Texas A&M University, College Station, TX.
Current molecular analysis is limited to use in central laboratory locations because it is bulky, costly, time-consuming, and requires users with considerable expertise in molecular biology to execute. This poster will describe an innovative nucleic acid analyzer and include data to demonstrate its utility in enabling an operator with minimal or no expertise in molecular biology to perform molecular diagnostics in both laboratory and non laboratory environment with improved speed and decreased instrumentation cost. Lynntech, in collaboration with Texas A&M University, is developing an incredibly simple, closed loop convective flow real-time PCR thermal cycler. The thermal cycler uses Rayleigh-Bénard convection to drive the liquid through three different temperature zones that enable denaturing, annealing, and extension steps commonly used in a PCR protocol. In our design, because the PCR mix is moved between fixed temperature zones, the need for dynamic thermal control is therefore eliminated. This significantly reduces the complexity of the level of hardware needed to run the device. In addition, the fixed temperature zones allow random-access and eliminate the need for sample batching. More importantly, rapid cycling times are achievable because reagents quickly attain thermal equilibrium with their surroundings as they are transported through successive temperature zones. This design is particularly attractive because unidirectional flow through the loop allows residence times within specific temperature zones to be precisely controlled for a particle assay condition. Moreover, the use of PCR tubing materials allows the user to monitor the PCR process in real-time with fluorescent dyes or probes. The versatility of the convective flow thermal cycling system was demonstrated by using it to amplify multiple targets (5 different respiratory viruses) with a wide product size range (1.3 kb to 191 bp). Our result also demonstrated that, given the same amount of PCR amplification duration, our cycler produced significantly more PCR products than a commercial cycler. We have also successfully demonstrated convective PCR amplification and detection of three species of Gram negative and Gram positive bacteria spiked in non-leukoreduced human blood platelets. Gel electrophoresis images of PCR products (16s rRNA gene) clearly demonstrated that the device is capable of detecting 100 CFU/mL bacteria (1 CFU/mL per PCR reaction) in the presence of excess human genomic materials from the platelets. We expect the sensitivity will be further improved when fluorescent detection set up is used to carry out real-time PCR reactions.In summary, we expect that our molecular analyzer can be adapted to run virtually any existing and future PCR or real-time PCR based clinical diagnostic assays. The ability to carry out rapid, near-patient molecular diagnostics will improve patient outcome and reduce the spread of highly infectious diseases. The device is specially designed for scenarios where the number of samples is relatively low and rapid turnaround is needed.
Poster 46A novel ELISA-based assay for the determination of symmetric dimethylarginine (SDMA). Pande R1, Padmanabhan M1, Murthy Y1, Atkinson M1, Nesbit S2, Fan T2, Yeung K1. 1IDEXX Laboratories, Westbrook, ME, and 2Beacon Analytical, Portland, ME.
Accurate assessment of renal function is an essential component of clinical diagnostics. Commonly used markers of renal function suffer from many drawbacks including lack of adequate response to early stage renal impairment. More sensitive markers of renal excretory function are needed, and recent studies (Nephrol Dial Transplant, 2006, 21, 2446-2451) indicate that symmetric dimethyl arginine (SDMA) could be a superior marker for assessing renal function. To facilitate further definitive studies to clarify whether SDMA is suited to improve kidney diagnosis, we have developed a simple quantitative immunoassay to detect SDMA from serum samples in contrast to the existing methods, such as HPLC, liquid chromatography-mass spectrometry (LC-MS) and gas chromatography (GC-MS). We generated highly specific SDMA antibodies and utilized these antibodies in a competitive ELISA. In the described assay, analyte samples are mixed and incubated with SDMA -Horseradish Peroxidase (SDMA-HRP) conjugate and SDMA antibody solution in a pre-coated 96 well microtiter plate. Following a wash step, and incubation with a colorimetric substrate, SDMA down to 0.04ug/mL can be detected. The assay is highly specific; with a cross-reactivity of <1%>300 dogs and cats) indicate that SDMA could be a useful marker for renal disease in dogs and cats.
Poster 47Digital microfluidics: a novel platform for multiplexing assays used in newborn screening.Rouse J1, Eckhardt AE 1, Millington DS2, Pamula VK1. 1Advanced Liquid Logic, RTP, NC, and 2Pediatrics, Duke University, Durham, NC.
Digital microfluidics using electrowetting is a powerful and flexible technique to manipulate discrete droplets independently and precisely under software control to perform biochemical assays on a disposable cartridge. We have demonstrated the utility of digital microfluidics in clinical, genomic, environmental, and other biochemical research applications. The objective of this work is to extend the platform to perform high-throughput newborn screening tests using dried blood spots (DBS). These assays currently require expensive equipment to process and analyze samples and consume significant amounts of sample. A digital microfluidic platform can greatly reduce assay costs and specimen requirements with an inexpensive instrument and test-specific disposable cartridges, potentially enabling screening at the point of birth. Using a disposable chip with the design shown in the figure below, multiplexed kinetic assays were developed for up to four enzymes, deficiencies of which result in lysosomal storage disease (LSD). Aqueous extracts from up to 12 DBS punches (3mm) and up to four enzyme substrates can be loaded on a chip and programmed to dispense, transport, mix, and incubate droplets of 300 nL volume. Enzymes in the samples cleave artificial fluorogenic substrates to release 4-methylumbelliferone (4-MU) which are measured on-chip using a custom-made miniature fluorescence detector (Ex-350nm/Em-420nm).
Poster 48Evaluation of Dynabeads® MyOne™ SILANE for purification of sequencing reactions. Berchanskiy D1, Ellis D2, Kalve I1, Dinauer D1. 1Life Technologies, Brown Deer, WI, and 2Invitrogen Dynal AS, Oslo, Norway.
Sequencing reaction clean-up is a crucial step in Sequence Based Typing procedures that renders accurate sequence analysis. Quality removal of unincorporated dyes and excess salts result in a higher signal and lower background. Here, we describe a performance comparison of Dynabeads SILANE with standard ethanol precipitation during sequencing clean-up. SILANE beads are magnetic particles that bind to DNA fragments thus allowing clean chromatographic separation and simultaneous removal of unincorporated dye terminators.The uniform and monodisperse SILANE beads are 1 μm in diameter and present a bead surface with optimized silica-like chemistry. An increased magnetic strength compared to other MyOne (1 µm) Dynabeads ensure rapid magnetic mobility and efficient capture of nucleic acids. The beads also feature a low sedimentation rate and favorable reaction kinetics, making them particularly suited for automated assays.To evaluate sequencing clean-up quality we used a variety of samples with Invitrogen's SeCore HLA-B and HLA-DRB1 SBT kits. A range of samples representing high to poor quality genomic DNA were amplified. After PCR clean-up and thermal cycling, sequencing reactions were pooled to ensure uniformity before performing sequencing clean-up with the two described methods. Sequencing results indicate that Dynabeads purification system is able to successfully remove unincorporated dyes and excess salts resulting in equivalent sequence quality and gaining higher signal intensity than ethanol precipitation. Use of Dynabeads reduced overall sample handling time by 15-20 minutes per 96-well plate.In conclusion, the Dynabeads sequencing clean-up method provides a good alternative to ethanol precipitation in terms of quality of sequence readout. Moreover, this simple to use system does not require centrifugation or filtration, which makes it particularly suitable to use with automated instrumentation ensuring minimal manual handling.
Poster 49Piezo-optical point-of-care immunoassay. Ross SA, Carter TJN. Vivacta Ltd, Kent Science Park, UK.
Piezoelectric polymer films, such as polyvinylidene fluoride (PVDF), respond to thermal and mechanical stress to generate a charge. One way of generating such a charge is by optical stimulation of a coloured agent adjacent to the polymer surface. Micro-heating causes stress in the PVDF, which can be measured electronically after amplification. This sensing system can be adapted for immunoassay applications by coating the surface of the PVDF with antibody and using a coloured label in a sample adjacent to the piezofilm. This technology is being developed by Vivacta as point-of-care product to monitor TSH levels in whole blood. In order to optimise the signal-to-noise ratio and reduce interference from red cells, a number of labels and illumination wavelengths have been tested. The best performance was found with 100 nm carbon particles illuminated with high intensity focussed 690 nm LEDs. The signal is monitored as a kinetic profile as the carbon particles diffuse to the surface of the sensor. There are no separation steps and the measurement can be made in whole blood, using a 30 µL sample in approximately 10 minutes or less. Analytical sensitivity for a sandwich assay measurement of a 30 kDa protein has been proven to 20 pg / mL in whole blood, although the system also works with serum or plasma samples.
Poster 51Development of a novel, universal real-time Transcription-Mediated Amplification based format for use in multiplex nucleic acid assays.Lyakhov DL, Carlson J, Nelson NC, Phelps SS, Chelliserrykattil J, Gordon PC, Kaminsky MB, Hashima SM, Ngo TV and Brentano ST. Gen-Probe Incorporated, San Diego, CA.
Use of multiplex amplified nucleic acid assays is gaining momentum in the clinical diagnostics arena. One drawback of such assays is the potential for adverse interactions between primer sets, resulting in decreased assay performance for one or more analytes in the mixture. The objective of the work presented here was to develop an assay format in which these adverse interactions were reduced or eliminated.Transcription-Mediated Amplification (TMA) is an isothermal amplification system that utilizes a non-T7 primer and a T7 primer/provider that contains a promoter for T7 RNA polymerase. In a standard multiplex TMA format, a specific primer/provider set is utilized for each analyte in the mixture throughout the course of the reaction.We have developed a new, universal real-time TMA format in which a single pair of universal sequence tags is incorporated into the specific NT7/T7 primer/provider pair for each analyte to be tested in a multiplex assay. Each of these analyte-specific NT7/T7 primer/provider pairs (all bearing the same 2 universal tags) is joined together to form a complex, and each complex is specifically bound to its corresponding analyte in the target capture phase of the assay. Excess complexes are washed away, amplification is initiated using the bound complexes, and a single universal primer/provider pair drives subsequent rounds of amplification for all analytes. No specific primers or providers are required in the amplification reaction, thus removing the potential for adverse interactions between primer/provider sets.In proof of principal studies (N=8), analytical sensitivity (≤ 100 copies/reaction) and precision (standard deviation ≤ 0.15 log copy number) of the assay were excellent for each analyte tested. The assay was quantitative (accuracy ± 0.2 log copy) with a wide dynamic range (≥ 5 logs). Furthermore, adverse primer/provider interactions were greatly reduced.In conclusion, a sensitive, accurate, and precise universal real-time TMA format that displays very low primer/provider interaction has been developed. This assay should show great utility for the simultaneous detection of multiple analytes in a variety of clinical diagnostic applications.