In an ever-changing biological environment, laboratory support is necessary to help responders identify, categorize, and manage incidents involving biological threats. The Pacific Northwest National Laboratory is one source that provides valuable testing data to help today’s first responders collect, screen,identify, and ultimately protect against such threats.
First responders know that white powder scenarios – or suspected biological threats – require quick and decisive action. Having the right field equipment available to identify suspicious substances can be complicated, challenging, and expensive. With support from the Department of Homeland Security (DHS) Science and Technology Directorate (S&T), Pacific Northwest National Laboratory (PNNL) has been working with the first responder community to identify biodetection technology and information needs and gaps, and transition solutions to the first responder community. PNNL’s “ground-up approach” involves first responders and stakeholders early in the process and culminates in the transition of information and knowledge to improve biological response capabilities (see Figure 1).
“Biodetection Technologies for First Responders: 2015 Edition”
Since 2012, PNNL has produced an annual product guide that has been downloaded almost 14,000 times. The guide summarizes commercially available technologies that can be used by first responders in the field for the collection, screening, and identification of suspected biothreat materials. This guide provides information about available technologies to help end users make informed decisions when procuring and using biodetection technology. The third edition, “Biodetection Technologies for First Responders: 2015 Edition” is available for download at: http://biodetectionresource.pnnl.gov (see Figure 2). The product guide is also available on the website in a search-and-compare format and as an iOS app (free from the Apple Store). Technology demonstration videos are also available on the website, as well as a demonstration of the American Society of Testing and Materials (ASTM) E2458-10 suspected biothreat powder collection standard practice.
PNNL tested 35 commercially available hand-portable biodetection products with over 5,000 assays conducted to date against anthrax, multiple levels of purity of ricin, and environmental powders. Product performance was evaluated in the areas of: inclusivity (ability to generate a true positive result for an actual biothreat sample); exclusivity (the ability to not generate a false positive result for a nonthreat sample); environmental interferents/powders (ability to not generate a false positive result or have assay interference); and sensitivity/limit of detection.
The products tested included biological indicator tests, immunoassays, and polymerase chain reaction (PCR) systems. Biological indicator tests simply detect the potential presence of biological material in a sample. Typically, these tests detect proteins, amino acids, deoxyribonucleic acid (DNA), or adenosine triphosphate, which are all indicators of a material of biological origin. These general biological screening tests detect a broad range of biological and organic materials, but do not confirm the presence of a specific biothreat agent. Therefore, although many of the biological indicator tests are relatively rapid and inexpensive, they should be used only as a screening tool in conjunction with tests that are more specific.
Agent-Specific Assays & Protein-Based Immunoassays
PNNL also tested agent-specific assays, which unlike the general biological indicators, can both detect and identify the specific agent or toxin present in a sample. PNNL tested both protein-based immunoassays and DNA-based PCR assays.
Immunoassays use antibodies, which are proteins designed (by nature or in the laboratory) to bind to a specific threat agent such as anthrax or ricin. Most field-based immunoassays use a lateral flow assay format similar to a home pregnancy test (see Figure 3). A lateral flow assay includes an assay strip containing all the assay components encased in a disposable plastic cartridge. The cartridge has a sample window where the sample is applied to the assay strip, and a results window where the results are visually displayed or read by an electronic optical reader. Immunoassays are advantageous because they are relatively inexpensive, require little skill to use, and results can typically be obtained in only 5 to 15 minutes. However, most immunoassays are several orders of magnitude less sensitive than PCR. Typical sensitivity for immunoassays range from 1 million to 10 million spores or microbes per milliliter.
In contrast to immunoassays, PCR-based assays detect specific organisms based on their DNA sequence. During PCR, short pieces of DNA from the biothreat organism are amplified, creating millions of DNA copies from a small number of starting molecules. PCR assays are designed to recognize regions of DNA that are unique to the biothreat organism. Most field-based PCR systems consist of a disposable assay cartridge containing all of the consumable reagents, an instrument that integrates the thermal components to perform the heating/cooling cycles required for PCR, and the optical components required for quantifying the amplified DNA products.
PCR assays are performed on liquid samples and require a sampling kit (sometimes included) to swab a suspicious powder and solubilize or suspend the powder in a compatible buffer. Depending on the system, various degrees of sample preparation or cartridge manipulation may be required, such as pipetting, manual mixing, or centrifugation. Results are visually displayed on the instrument following the PCR reaction. PCR-based assays are advantageous because they are very sensitive and specific (although the specificity of a system is dependent on the design of a particular assay).
Very few field-based PCR systems have integrated sample preparation to concentrate DNA and remove PCR inhibitors. However, because PCR is very sensitive, a sample can often be significantly diluted after sampling to reduce the effects of potential inhibitors on the reaction. Few published studies are available that assess the impact of environmental samples and hoax powders on PCR-based assays when little or no sample preparation is conducted. The most significant disadvantages of PCR-based approaches are relatively long assay times (typically 30 to 60 minutes) and, for some systems, relatively high costs (see Figure 4).
A Test Plan, Gaps & New Products
Prior to testing, PNNL developed a statistically based, cost-effective test plan that provides results for the probability of detection and percent confidence to define the performance of a given biodetection product. This test plan has formed the basis for a proposed ASTM standard in the E54.01 Homeland Security subgroup.
However, serious gaps still exist in the first responder biodetection community including a lack of: (a) test and evaluation standards; (b) formal guidance for conducting field biodetection; (c) biodetection product performance data obtained by an independent third party; (d) best practices and knowledge for technology use and limitations; and (e) a consolidated resource for biodetection-related information.
New, untested biodetection products continue to emerge at a rapid pace, numerous other biothreat agents have not been tested on any platforms, and additional testing with more complex types of samples and a broader range of “near-neighbor” agents (nonpathogenic organisms similar to biothreat organisms) remains to be done. While it is critical (and more cost-effective) to demonstrate instrument performance and proficiency in the laboratory, it is equally important for first responders to evaluate instruments in the field, after the instruments have successfully passed the laboratory testing.
In addition, a sustained effort is needed to transition information to the first responder community to keep responders informed of performance testing results, technology use and limitations, and best practices. Conducting and supporting bioresponse field exercises that promote coordination between first responders, the Federal Bureau of Investigation (FBI), public health/Laboratory Response Network labs, and National Guard Bureau Civil Support Teams are also critical to identify gaps and improve response readiness and effectiveness. Although PNNL efforts are bridging these gaps to improve overall first responder effectiveness and public safety, considerable work remains to be done.
Significant contribution to this article was made by Rachel A. Bartholomew, Ph.D., is a senior research scientist at Pacific Northwest National Laboratory and has over 15 years of experience in molecular biology, including developing and testing systems for environmental biodetection, cell culture and diagnostics, and national security applications. She has publications in the area of biodetection, cell culture, and molecular biology, including an upcoming chapter on polymerase chain reaction (PCR) in the American Society of Microbiology’s publication “Methods in Environmental Microbiology” (4th edition). She received an undergraduate degree in biology from Case Western Reserve University and a Ph.D. in physiology from Cornell University.
Richard M. Ozanich, Ph.D., has worked in the chemical and biodetection fields for over 25 years. He is a subject matter expert in biodetection and optical spectroscopy with a broad base of knowledge in chemistry, biology, and measurement instrumentation. He is active in the area of bioresponse and development of standards and best practices and is a member of American Society for Testing and Materials Committee E54 on Homeland Security Applications. His research includes development of automated fluidics instrumentation and microparticle-based methods for sample preparation and rapid detection of biothreats.
Cynthia J. Bruckner-Lea
Cynthia J. Bruckner-Lea, Ph.D., is a senior scientist and program manager at Pacific Northwest National Laboratory. She is a recognized biodetection expert with over 30 years of experience in the development and application of biological detection systems for environmental monitoring, medical, and national security applications. She is an American Association for the Advancement of Science Engineering Section Fellow, and she has served on several National Academy of Science Committees conducting studies related to chemical and biological detection. She has over 50 publications and 10 patents. She received a B.S. degree in chemical engineering from the University of California, Davis, and a Ph.D. in bioengineering from the University of Utah.