Acceptance Criteria Framework for Autonomous Biological Detectors

The purpose of this study was to examine a set of user acceptance criteria for autonomous biological detection systems for application in high-traffic, public facilities. The test case for the acceptance criteria was the Autonomous Pathogen Detection System (APDS) operating in high-traffic facilities in New York City (NYC). However, the acceptance criteria were designed to be generally applicable to other biological detection systems in other locations. For such detection systems, ''users'' will include local authorities (e.g., facility operators, public health officials, and law enforcement personnel) and national authorities [including personnel from the Department of Homeland Security (DHS), the BioWatch Program, the Centers for Disease Control and Prevention (CDC), and the Federal Bureau of Investigation (FBI)]. The panel members brought expertise from a broad range of backgrounds to complete this picture. The goals of this document are: (1) To serve as informal guidance for users in considering the benefits and costs of these systems. (2) To serve as informal guidance for developers in understanding the needs of users. In follow-up work, this framework will be used to systematically document the APDS for appropriateness and readiness for use in NYC

Development of an automated DNA purification module using a micro-fabricated pillar chip

We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 &times; 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 &micro;m, respectively, which provides a relatively large surface area (ca. 3 cm2) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.<br /

Fielding the NIF Cryogenic Ignition Target

The United States Department of Energy has embarked on a campaign to conduct credible fusion ignition experiments on the National Ignition Facility (NIF) at the Lawrence Livermore National Laboratory in 2010. The target assembly specified for this campaign requires the formation of a deuterium/tritium (DT) fuel ice layer on the inside of a 2 millimeter diameter capsule positioned at the center of a 9 millimeter long by 5 millimeter diameter cylinder, called a hohlraum. The ice layer requires micrometer level accuracy and must be formed and maintained at temperatures below 19 K. At NIF shot time, the target must be positioned at the center of the NIF 10 meter diameter target chamber, aligned to the laser beam lines and held stable to less than 7 micrometers rms. We have completed the final design and are integrating the systems necessary to create, characterize and field the cryogenic target for ignition experiments. These designs, with emphasis on the challenges of fielding a precision cryogenic positioning system will be presented

Hybridization and Selective Release of DNA Microarrays

DNA microarrays contain sequence specific probes arrayed in distinct spots numbering from 10,000 to over 1,000,000, depending on the platform. This tremendous degree of multiplexing gives microarrays great potential for environmental background sampling, broad-spectrum clinical monitoring, and continuous biological threat detection. In practice, their use in these applications is not common due to limited information content, long processing times, and high cost. The work focused on characterizing the phenomena of microarray hybridization and selective release that will allow these limitations to be addressed. This will revolutionize the ways that microarrays can be used for LLNL's Global Security missions. The goals of this project were two-fold: automated faster hybridizations and selective release of hybridized features. The first study area involves hybridization kinetics and mass-transfer effects. the standard hybridization protocol uses an overnight incubation to achieve the best possible signal for any sample type, as well as for convenience in manual processing. There is potential to significantly shorten this time based on better understanding and control of the rate-limiting processes and knowledge of the progress of the hybridization. In the hybridization work, a custom microarray flow cell was used to manipulate the chemical and thermal environment of the array and autonomously image the changes over time during hybridization. The second study area is selective release. Microarrays easily generate hybridization patterns and signatures, but there is still an unmet need for methodologies enabling rapid and selective analysis of these patterns and signatures. Detailed analysis of individual spots by subsequent sequencing could potentially yield significant information for rapidly mutating and emerging (or deliberately engineered) pathogens. In the selective release work, optical energy deposition with coherent light quickly provides the thermal energy to single spots to release hybridized DNA. This work leverages LLNL expertise in optics, microfluids, and bioinformatics

LAVA: An Open-Source Approach To Designing LAMP (Loop-Mediated Isothermal Amplification) DNA Signatures

<p>Abstract</p> <p>Background</p> <p>We developed an extendable open-source Loop-mediated isothermal AMPlification (LAMP) signature design program called LAVA (LAMP Assay Versatile Analysis). LAVA was created in response to limitations of existing LAMP signature programs.</p> <p>Results</p> <p>LAVA identifies combinations of six primer regions for basic LAMP signatures, or combinations of eight primer regions for LAMP signatures with loop primers, which can be used as LAMP signatures. The identified primers are conserved among target organism sequences. Primer combinations are optimized based on lengths, melting temperatures, and spacing among primer sites. We compare LAMP signature candidates for <it>Staphylococcus aureus </it>created both by LAVA and by PrimerExplorer. We also include signatures from a sample run targeting all strains of <it>Mycobacterium tuberculosis</it>.</p> <p>Conclusions</p> <p>We have designed and demonstrated new software for identifying signature candidates appropriate for LAMP assays. The software is available for download at <url>http://lava-dna.googlecode.com/</url>.</p

Stability Measurements for Alignment of the NIF Neutron Imaging System Pinhole Array

The alignment system for the National Ignition Facility's neutron imaging system has been commissioned and measurements of the relative stability of the 90-315 DIM, the front and the back of the neutron imaging pinhole array and an exploding pusher target have been made using the 90-135 and the 90-258 opposite port alignment systems. Additionally, a laser beam shot from the neutron-imaging Annex and reflected from a mirror at the back of the pinhole array was used to monitor the pointing of the pinhole. Over a twelve hour period, the relative stability of these parts was found to be within {approx} {+-}18 {micro}m rms even when using manual methods for tracking the position of the objects. For highly visible features, use of basic particle tracking techniques found that the front of the pinhole array was stable relative to the 90-135 opposite port alignment camera to within {+-}3.4 {micro}m rms. Reregistration, however, of the opposite port alignment systems themselves using the target alignment sensor was found to change the expected position of target chamber center by up to 194 {micro}m

Draft Guidance: Response, Restoration, and Recovery Checklist for Biologically Contaminated Facilities

The Checklist for Facility Response, Restoration, and Recovery presented in this document is principally focused on the Consequence Management Phase of a biothreat agent (i.e., Bacillus anthracis) release at a large facility, such as an airport or subway. Information in this document conforms to the National Response Plan (NRP) (DHS 2004) and the National Incident Management System (NIMS 2004). Under these two guidance documents, the personnel responsible for managing biological response and recovery efforts--that is, the decision-makers--are members of an Incident Command (IC), which is likely to transition to a Unified Command (UC) in the event of a biological warfare agent attack. A UC is used when more than one agency has incident jurisdiction or when incidents cross political jurisdictions. The location for primary, tactical-level command and management is referred to as the Incident Command Post (ICP), as described in the NRP. Thus, regardless of whether an IC or an UC is used, the responsible entities are located at an ICP. Agencies work together through designated members of the UC to establish their designated Incident Commanders at a single ICP and to establish a common set of objectives and strategies and a single Incident Action Plan. Initially during the Crisis Management Phase, the Incident Commander is likely to be the Chief of the fire department that serves the affected facility. As life-safety issues are resolved and the Crisis Management Phase shifts to the Consequence Management Phase, the work of characterization, decontamination, and facility clearance begins. There will likely be a coincident transition in organizational structure as well, and new restoration-focused groups, units, and personnel will be added as restoration needs are anticipated. Depending on the specific facility and type of incident, the responsible individual (Incident Commander or Unified Commander) within the UC during the Consequence Management Phase could be the Facility Manager, the Facility Emergency Operations Manager, or their designee. In an incident involving large-scale biological contamination, the Governor of the state would typically request, and the President of the United States would likely declare, an emergency under the Stafford Act (1974; amended 2002). The Secretary of Homeland Security would likely determine that the event is an Incident of National Significance on the basis of criteria established in Homeland Security Presidential Directive 5 (HSPD-5), ''Management of Domestic Incidents''. Incidents of National Significance are those high-impact events that require a coordinated and effective response by an appropriate combination of Federal, state, local, tribal, private-sector, and nongovernmental entities to save lives, minimize damage, and provide the basis for long-term community recovery and mitigation activities. If facility authorities request outside assistance, or if an emergency is declared under the Stafford Act, then other members of the UC could include local and state agencies as well as Federal agencies, such as the Federal Emergency Management Agency (FEMA) and the U.S. Environmental Protection Agency (USEPA). If an Incident of National Significance is declared, a Principal Federal Official will be appointed by the Department of Homeland Security (DHS) to facilitate Federal support to the UC structure. The following Checklist for Facility Response, Restoration, and Recovery presents the critical steps that would be taken by organizations involved in responding to a biological incident. It is intended for use by key decision-makers in the event that an incident occurs and steps must be taken immediately and systematically. The organizations would follow the Incident Command System (ICS). See Appendix A for more information on the ICS and how the responsible personnel identified in the checklist map into the consequence management organizational structure. The Notification and First-Response Phases are cursorily addressed in the checklist, whereas the main focus is on consequence management actions. The order of actions is generally sequential. However, depending on the specifics of an event and how the response is implemented, actions may be reordered. For example, preparing a Remediation Action Plan (RAP) is identified in the checklist as a critical step of the Remediation Phase. However, it is likely that preparation of the RAP would begin before completing all actions identified in the Characterization Phase. In addition to the actions recommended in the checklist, any emergency response conducted at a major metropolitan facility should comply with notification and response procedures established by the facility, as well as applicable procedures established by the jurisdictional responding agencies

Lessons from Two Years of Building Fusion Ignition Targets with the Precision Robotic Assembly Machine

The Precision Robotic Assembly Machine was developed to manufacture the small and intricate laser-driven fusion ignition targets that are being used in the world's largest and most energetic laser, the National Ignition Facility (NIF). The National Ignition Campaign (NIC) goal of using the NIF to produce a self-sustaining nuclear fusion burn with energy gain - for the first time ever in a laboratory setting - requires targets that are demanding in materials fabrication, machining, and assembly. We provide an overview of the design and function of the machine, with emphasis on the aspects that revolutionized how NIC targets are manufactured

Performance Improvements to the Neutron Imaging System at the National Ignition Facility

A team headed by LANL and including many members from LLNL and NSTec LO and NSTec LAO fielded a neutron imaging system (NIS) at the National Ignition Facility at the start of 2011. The NIS consists of a pinhole array that is located 32.5 cm from the source and that creates an image of the source in a segmented scintillator 28 m from the source. The scintillator is viewed by two gated, optical imaging systems: one that is fiber coupled, and one that is lens coupled. While there are a number of other pieces to the system related to pinhole alignment, collimation, shielding and data acquisition, those pieces are discussed elsewhere and are not relevant here. The system is operational and has successfully obtained data on more that ten imaging shots. This remainder of this whitepaper is divided in five main sections. In Section II, we identify three critical areas of improvement that we believe should be pursued to improve the performance of the system for future experiments: spatial resolution, temporal response and signal-to-noise ratio. In Section III, we discuss technologies that could be used to improve these critical performance areas. In Section IV, we describe a path to evolve the current system to achieve improved performance with minimal impact on the ability of the system to operate on shots. In Section V, we discuss the abilities, scope and timescales of the current teams and the Commissariat energie atomique (CEA). In Section VI, we summarize and make specific recommendations for collaboration on improvements to the NIS