Five-Step Feild-Test Process For Ruling Out Anthrax Spores

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Following the anthrax mail attack on the eastern United States in September 2001, emergency response organizations across the country were inundated with “white-powder” incidents. All but a few of the calls, related to the actual anthrax attack on the East Coast, were hoaxes.

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Photo By Robert Burke
During training at the Center for Domestic Preparedness (CDP) in Anniston, AL, an emergency responder in Level C protective clothing uses a flashlight to better view turbidity in a test sample.

Responding to the extremely high number of white-powder incidents taxed the resources of many response organizations. There were so many requests for assistance that the FBI could not respond to all of them; the bureau wanted to be notified only of “credible” incidents. Analytical laboratories that were asked to identify samples from incidents were overwhelmed by the number of requests. Field tests available at the time to test for anthrax spores were unreliable and resulted in many false positives. Lab test equipment that could be used in the field to test for anthrax is very expensive and not available in most areas outside the military.

Personnel at the Center for Domestic Preparedness (CDP) in Anniston, AL, saw the need for a better way to deal with white-powder incidents. CDP, a component of the Office for Domestic Preparedness, is the U.S. Department of Homeland Security training center for weapons of mass destruction (WMD). It is the only emergency responder training center in the world that provides live-agent training using sarin and VX nerve agents. The center opened in 1998 and has trained 186,000 emergency responders through on- and off-campus training opportunities. The facilities are situated on 62 acres at the site of a former Army base, Fort McClellan. Plans are in the works to acquire an additional 23.5 acres at the fort with buildings that will be used for hands-on training. In addition, CDP conducts WMD equipment evaluation using first responders to determine the usefulness of various items on the market. Equipment is obtained from manufacturers and emergency responders assess them under realistic conditions. Responders wear full personal protective equipment (PPE) when appropriate. While I was there, responders were evaluating equipment for carrying non-ambulatory patients out of incident hot zones, through decontamination, and on to triage and treatment.

Table 1: Proposed Biological Field Test System
Test to be Conducted
Possibly, a biological (or of respirable size)*
Not Likely to be biological (or not of respirable size)*
Step 1. Collect two samples: Sample 1 for reference laboratory analysis**; a volume equivalent to one restaurant sugar packet (ca. 1 gm) or more, if available. Sample 2 for field analysis; a volume similar to the size of a small pea or kernel of corn.


Step 2. Place material to be analyzed in a dry ca. 3ml clean glass vial and secure lid. Shake vigorously for a few seconds and observe.
Fine cloud or haze hangs above sample for several seconds after shaking is stopped.
All material falls to bottom of vial, like salt in a salt shaker, after shaking; air above material is clear.
Step 3. Remove lid, fill vial ca two-thirds (ca. 2 ml) with distilled water and resecure lid. Shake vigorously for 15 seconds and observe.
Sample appears to mix with water, but does not dissolve. Liquid contents remain turbid or cloudy.
Sample dissolves in water and becomes clear with or without larger particles settling to the bottom.
Step 4. Remove lid of vial and dip pH test strip into water. Remove strip, wait 30 seconds and read result on pH strip container.
pH between 5 and 9
pH less than 5 or greater than 9
Step 5. Remove lid of vial and dip one protein test strip. Remove strip, wait 30 seconds and read result on protein strip container.
Protein is present.
Protein is not present.
Note: *The shaking of dry powder in a vial as described in Step 2 provides only an indication of particle size. This part of the test protocol does not provide an indication regarding the potential for the material being of biological origin.
** Standing procedures that address packaging , chain of custody and decontamination for suspect samples should be established with the local FBI in advance of an event.
Source: Homeland First Response, July/August 2003

To deal with problems associated with white-powder incidents, CDP assembled a working group in December 2002 to develop a protocol for use by emergency responders at incident scenes. The goal was to pursue a low-cost way to detect anthrax spores in the field. Members of the working group included scientists, representatives from the fire service, law enforcement, EMS and hazardous materials response organizations, and emergency response educators. An analysis of responses to white-powder/biological-agent incidents yielded weaknesses in preparedness at the local level:

  • Lack of standardized protocols for response to biological incidents.
  • Large numbers of responses exceeded local capabilities.
  • Mid-sized and small communities lacked means of analysis to rapidly screen materials in the field to differentiate between potential hoax materials and real biological materials.
  • Lack of safety guidelines for response to white-powder incidents resulting in both over- reaction and under-reaction.
  • Confusing and conflicting information from emergency responders resulted in skepticism among members of the public.
  • Some detection equipment sold on the market is unregulated and invalidated.

Beginning the problem-solving process the working group established two objectives:

2. Develop a consensus field analysis protocol for responding to a potential anthrax letter.

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Photo By Robert Burke
A CDP student using a test strip to check for protein content compares his findings to a container comparison chart. Determining protein content helps emergency responders evaluate suspect powders.

By the summer of 2003, as a result of the working group’s efforts, a five-step biological field test system was proposed. First of all, it is important to note that the field test does not specifically identify anthrax spores or any other biological agent. What the field test system does is to “rule out” the presence of biological materials in a sample from a suspected white powder or biological agent.

Several factors regarding biological agents were addressed in the development of the testing system. First, biological agents are living organisms that typically survive and multiply within a pH range of 6.5 to 8.0. They contain protein and other components such as carbohydrates, fats and nucleic acids (DNA and RNA). Most biological materials, including anthrax spores, create a turbid (opaque or muddy) suspension when placed in water. Based on the natural characteristics known about potential biological materials, the working group established test procedures designed to “rule out” the presence of biological agents. These procedures focus on particle size, water solubility, pH and protein content to assist emergency responders in evaluating unknown suspect powders. The Five-Step Testing Process is presented in Table 1.

The Five-Step Process is designed to be simple to conduct with minimal equipment. Supplies for the test kit include three-milliliter glass or borosilicate vials; pH paper test strips with a container comparison chart; protein test strips with a container comparison chart; a small disposable spatula or scoop; distilled water; a magnifying glass (10 or 20x); and a flashlight. A minimum of Level C personal protective clothing and respiratory protection with appropriate filters needs to be worn during sampling and testing procedures.

Tests should be performed at the site where the powder or container of powder is located (within the “hot zone”) so that contamination is not spread. This testing process does not eliminate the need to take a sample or samples to be sent out for laboratory analysis, so in addition to the test sample, response personnel should have the necessary supplies for collecting a sample to go to the lab. Labs should have been contacted in advance of an incident to determine if they can test for biological materials and what they require in the way of sample collection, preservation, and sample size. Standing procedures that address packaging, chain of custody and decontamination for suspect samples should be established with the local FBI or other law enforcement agencies in advance of an event.

Biological experts have validated the effectiveness of the field-test procedures in the laboratory with a confidence level of 99%. That is to say if all of the steps in the Five-Step Process produce negative results, there is a 99% chance that there is no anthrax or other biological agent present. Live anthrax spores were used to test the effectiveness of the test kit in the laboratory setting.

CDP has developed a chart titled Simple Biological Field Test System Confidence Factors to help response personnel interpret unclear test results and make decisions regarding powder incidents (see Table 2). The shake test is used to determine particle size. Solubility, pH and protein test results, no matter in what order conducted, can be evaluated by comparing results to Table 2. This will ascertain the approximate likelihood that the sample does not contain anthrax spores. Test scores that are in the red zone are considered to be dangerous and should be treated as such. Those in the amber and yellow zones should also be considered potentially dangerous. Green zone scores are considered very unlikely to be a biological threat, and may be treated as such.

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Photo By Robert Burke
Chemical/biological (CB) personal protective equipment kits can be pre-packaged for quick use by emergency responders when conducting on-scene testing of samples.

Once the working group reported its findings and the procedures were validated by biological experts the next step was to determine how effectively emergency response personnel could utilize the procedure in the field. I was invited by CDP to witness the testing process and prepare this article for Firehouse® to get the word out regarding this revolutionary new testing procedure.

A group of hazardous materials technicians attending CDP the week of June 21-25, 2004, were asked to stay an extra two days following their terrorism training class to perform operational tests using the Five-Step Process. Responders were provided with an overview of the test-development process, instructions for conducting the tests, test kits and personal protective equipment. Responders were divided into pairs and unmarked test samples were provided from the 15 most common substances used in white-powder incidents. Harmless biological materials were also present among the test samples to show positive results from the testing process. For security purposes those materials will not be identified here.

The testing apparatus and procedures did not appear to cause any significant difficulty for response personnel. Each group of responders conducted tests on some of the 15 samples without protective equipment to become familiar with the procedures. Each responder was then given Level C protective clothing, a cartridge respirator, protective booties and two layers of gloves. An additional layer of latex gloves was placed on the outside of the butyl rubber gloves to make it easier to handle the test equipment and materials with gloved hands. Once again there did not appear to be any major problems with response personnel conducting the tests using full Level C personal protective equipment.

The complete test was conducted in a much shorter time than predicted by CDC staff. Results of the operational testing conducted by the emergency responders will be evaluated by scientists at the Aberdeen Proving Ground in Maryland. Initial findings from the laboratory and operational testing indicate the process is effective at ruling out anthrax specifically and other biological agents generally. Further tests are ongoing to ensure that the best methods of employing the entire process are developed and documented prior to release of the final reports.

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Robert Burke, a Firehouse® contributing editor, is the fire marshal for the University of Maryland. He is a Certified Fire Protection Specialist (CFSP), Fire Inspector II, Fire Inspector III, Fire Investigator and Hazardous Materials Specialist, and has served on state and county hazardous materials response teams. Burke is a veteran of 24 years in fire and emergency services, with experience in career and volunteer departments. He has attained the rank of lieutenant, assistant chief and deputy state fire marshal. Burke is an adjunct instructor at the National Fire Academy and the Community College of Baltimore, Catonsville Campus, and the author of the textbooks Hazardous Materials Chemistry for Emergency Responders and Counter Terrorism for Emergency Responders. He can be reached via the Internet at robert.burke@att.net.

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