In October 2001, an American Media Incorporated employee died from inhalation anthrax disease. In the same month, contaminated letters laced with Bacillus anthracis, or anthrax spores, were sent through the mail to Senators Thomas Daschle and Patrick Leahy. The response to the incident in the American Media Incorporated building in Florida in September 2001 led to the identification of mail as the potential source of contamination; eventually, it led to the sampling of the postal facilities. The agencies began sampling on October 12, 2001, in Florida and stopped on April 21, 2002, when the Wallingford, Connecticut, facility was sampled for the last time. The letters led to the first cases of anthrax disease related to bioterrorism in the United States. In all, 22 individuals contracted anthrax disease in Connecticut, Florida, New Jersey, and New York, as well as in Washington, D.C., and 5 died. The federal agencies involved in the response in the postal facilities have different responsibilities. CDC and state and local health departments primarily provided public health advice and assistance to USPS. CDC has primary responsibility for national surveillance of specific diseases, including anthrax; it also conducts epidemiologic investigations to determine, among other things, the source of the disease, and it participates in environmental sample collection and analysis activities. The FBI is responsible for criminal investigations involving interstate commerce and the mail and crimes committed on federal property. EPA is the nation’s lead agency for responding to a release of hazardous substances into the environment and subsequent decontamination. On October 8, 2001, the President created the Office of Homeland Security to develop and coordinate a comprehensive national strategy for dealing with domestic terrorist threats or attacks. The office, which had limited involvement in the 2001 response, was superseded by the Homeland Security Act of 2002, which transferred many of its functions to DHS, which became operational in 2003. DHS was created by combining many previously separate agencies and is assigned a lead role in coordinating the efforts of federal agencies that respond to acts of terrorism in the United States. The federal agencies primarily used a targeted strategy—they collected samples from specific areas considered more likely to be contaminated, based on judgments. Such judgments can be effective in some situations— for example, in determining whether a facility is contaminated when information on the source of potential contamination is definitive. However, in the case of a negative finding, when the source of potential contamination is not definitive, the basic question—Is this building contaminated?—will remain unanswered. CDC and USPS officials said that they used a targeted strategy for several reasons, including limitations on how many samples could be collected and analyzed. They also said that in 2001, they lacked the data from empirical research to develop an initial sampling strategy that incorporated probability sampling. We disagree with this interpretation. Probability sampling is statistically based and does not depend solely on empirical criteria regarding the details of possible contamination. The situation in 2001 was unique, and the agencies were not fully prepared to deal with environmental contamination. In the future, if the agencies decide to use a targeted rather than a probability sampling strategy, they must recognize that they could lose a number of days if their targeted sampling produces negative test results. In this case, additional samples would have to be collected and analyzed, resulting in the loss of critical time for public health interventions. This was so at the Wallingford postal facility in the fall of 2001, when about 3 weeks elapsed between the time the first sampling took place and the results of the fourth testing, which revealed positive results. Furthermore, about 5 months elapsed between the time of the first sampling event and the time anthrax was found in the Wallingford facility’s high-bay area. Therefore, strategies that include probability sampling need to be developed in order to provide statistical confidence in negative results. Further, even if information on all the performance characteristics of methods is not yet available, a probability sampling strategy could be developed from assumptions about the efficiency of some of the methods. And even if precise data are not available, a conservative, approximate number could be used for developing a sampling strategy. This would give agencies and the public greater confidence in negative test results than was associated with the sampling strategy used in 2001. CDC, EPA, and USPS, the federal agencies involved in sampling the postal facilities in 2001 to detect anthrax, undertook several activities. These included development of a sampling strategy followed by collecting samples, using a variety of methods, and transporting, extracting, and analyzing the samples. Neither these activities nor the overall process was validated for anthrax testing. Consequently, the agencies were challenged by limited information for reliably choosing one method over another and by lack of information on the detection limit to use when evaluating negative results. Federal agencies used different methods for collecting samples. While USPS generally used dry swabs to collect samples (the least effective method), CDC and EPA used multiple methods—dry swabs, premoistened swabs, wet wipes, and a high-efficiency particulate air (HEPA) vacuum— in various combinations or alone. However, none of the agencies’ collection methods were evaluated for anthrax detection in environmental samples. In the absence of empirical research, agencies had no information available for reliably choosing one method over another and no information on the limits of detection to use when evaluating negative results. The majority of the samples collected from the postal facilities tested negative. In all, federal agencies collected about 10,000 samples during initial testing. It is interesting that of the 9,807 samples that the agencies collected, more than 98 percent, or 9,648, were negative; a little more than 1 percent, or 159, were positive. In all, 286 facilities were tested for anthrax contamination. Of these, Brentwood, Trenton, and Morgan were primary facilities; that is, these 3 facilities processed the original letters containing the anthrax. The results of the CDC, EPA, and USPS testing in 286 postal facilities were largely negative. Of 286 facilities, 23 tested positive. For 2 of these 23 facilities, test results were negative at first but positive on a subsequent testing. However, in 1 of these facilities—the Wallingford, Connecticut, facility—it was not until the fourth testing that positive results were obtained. Testing results differed between the primary facilities and Wallingford. In the 3 primary facilities, results were positive each time a facility was tested, with the important exception of the two quick tests in Brentwood. In Wallingford, considered less likely to be contaminated, results were positive only on the fourth sampling. These results underscore the importance of retesting and cast doubt on the efficiency of the judgmental sampling strategy. Of the 263 facilities that tested negative, only 9 were sampled more than once. A facility in West Trenton tested negative, even though an employee had contracted cutaneous anthrax. The facility in West Trenton was tested twice by the FBI and once by CDC, during which a total of 57 samples were collected, with negative results. Final, or confirmed, results will be negative if contamination is not present in a facility. However, a result can be erroneously negative for several other reasons, such as (1) the sampling method was not efficient enough, (2) samples were not collected from places where contamination was present, (3) not enough samples were collected, (4) not enough spores were recovered from the sample material, or (5) analysis of the sample extract was not sensitive enough to detect anthrax spores that were present. The agencies that sampled postal facilities in 2001—USPS, CDC, and EPA—did not use validated sample collection and analysis methods to perform their tests. According to these agencies, validated methods were not available at that time. They conducted several interdependent activities, including sample strategy development, followed by sample collection, transportation, and analysis of the samples to detect anthrax. Neither these activities nor the overall process had been validated for anthrax testing. Validation is a formal, empirical process in which an authority determines and certifies the performance characteristics of a given method. Therefore, investments are also needed to validate these methods, as well as the overall anthrax detection process. Validating the overall process, as well as the individual activities, is important because operational and health-related decisions are made on the basis of testing results that the process generates. CDC and USPS officials said that they used targeted sampling; that is, they collected samples from specific areas considered—based on agencies’ technical judgments—more likely to be contaminated. Such judgments can be effective in some situations, for example, in determining the source of contamination in a disease outbreak investigation, provided results are positive. However, if the results are negative, the basic question—Is this building contaminated?—cannot be answered with statistical confidence. When the level of contamination is extremely high and dispersed in a facility, the method of sampling (for example, wipes versus swabs) is not as critical if the purpose is to find some contaminant. However, at lower levels, a way of interpreting the significance of negative results is needed, and this requirement emphasizes the importance of validation of the methods and the need for statistically based sampling strategies. This emphasizes the need for methods that have been validated, and sampling strategies that are likely to find contamination at low levels. Probability- based sampling does allow conclusions, at specific levels of confidence, about testing results. Using a probability-based sampling strategy, together with validated methods for detecting contamination, would provide a known level of confidence with which to interpret any negative results. This would allow agencies to be more definitive in determining necessary actions. Figure 1 shows how lack of validation could affect individual activities—including the sampling strategy—as well as the results generated by the overall process. The lack of validated methods for assessing contamination in postal facilities impeded the agencies in responding to the incidents. The significance of the lack of validated methods was exemplified in the case of the one postal facility where negative preliminary results were obtained by field-based methods of analysis, with limitations that appear not to have been well understood by some agencies.