Summary

Less than a month after the September 11, 2001, attacks, letters containing spores of anthrax bacteria (Bacillus anthracis, or B. anthracis) were sent through the U.S. mail. Between October 4 and November 20, 2001, 22 individuals developed anthrax; 5 of the cases were fatal.

Although it was initiated as a public health investigation, the investigation quickly fell under the purview of the Federal Bureau of Investigation (FBI), when a deliberate act was suspected and letters containing B. anthracis were discovered in both New York and Washington, D.C., addressed to Tom Brokaw of NBC News, the New York Post, and U.S. Senators Tom Daschle of South Dakota and Patrick Leahy of Vermont.

Over the course of its investigation, the FBI devoted 600,000 investigator work hours to the case and assigned 17 Special Agents to a Task Force, along with 10 U.S. Postal Inspectors. The investigation spanned six continents; involved over 10,000 witness interviews, 80 searches, 26,000 email reviews, and analyses of 4 million megabytes of computer memory; and resulted in the issuance of 5,750 grand jury subpoenas. Additionally, 29 government, university, and commercial laboratories assisted in conducting the scientific analyses that were a central aspect of the investigation (U.S. Department of Justice [USDOJ], 2010, p. 4).

During its investigation of the anthrax mailings, the FBI worked with other federal agencies to coordinate and conduct scientific analyses of the anthrax letter spore powders, environmental samples, clinical samples, and samples collected from laboratories that might have been the source of the letter-associated spores. The agency relied on external experts, including some who had previously developed tests to differentiate among strains of B. anthracis.

In 2008, seven years into the investigation, the FBI asked the National Research Council (NRC) of the National Academy of Sciences (NAS) to conduct an independent review of the scientific approaches used during the investigation of the 2001 B. anthracis mailings (see Box S-1 for charge). In addition to informing FBI investigators about possible leads, much of the science used



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Summary Less than a month after the September 11, 2001, attacks, letters containing spores of anthrax bacteria (Bacillus anthracis, or B. anthracis) were sent through the U.S. mail. Between October 4 and November 20, 2001, 22 individuals developed anthrax; 5 of the cases were fatal. Although it was initiated as a public health investigation, the investigation quickly fell under the purview of the Federal Bureau of Investigation (FBI), when a deliberate act was suspected and letters containing B. anthracis were discovered in both New York and Washington, D.C., addressed to Tom Brokaw of NBC News, the New York Post, and U.S. Senators Tom Daschle of South Dakota and Patrick Leahy of Vermont. Over the course of its investigation, the FBI devoted 600,000 investiga- tor work hours to the case and assigned 17 Special Agents to a Task Force, along with 10 U.S. Postal Inspectors. The investigation spanned six continents; involved over 10,000 witness interviews, 80 searches, 26,000 email reviews, and analyses of 4 million megabytes of computer memory; and resulted in the issu- ance of 5,750 grand jury subpoenas. Additionally, 29 government, university, and commercial laboratories assisted in conducting the scientific analyses that were a central aspect of the investigation (U.S. Department of Justice [USDOJ], 2010, p. 4). During its investigation of the anthrax mailings, the FBI worked with other federal agencies to coordinate and conduct scientific analyses of the anthrax letter spore powders, environmental samples, clinical samples, and samples col- lected from laboratories that might have been the source of the letter-associated spores. The agency relied on external experts, including some who had previ - ously developed tests to differentiate among strains of B. anthracis. In 2008, seven years into the investigation, the FBI asked the National Research Council (NRC) of the National Academy of Sciences (NAS) to con- duct an independent review of the scientific approaches used during the inves - tigation of the 2001 B. anthracis mailings (see Box S-1 for charge). In addition to informing FBI investigators about possible leads, much of the science used 1

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2 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS BOX S-1 Charge to the Committee The NRC was asked by the FBI to conduct an independent review of the scientific approaches used during the investigation. The official charge to the committee stated: An ad hoc committee with relevant expertise will evaluate the scientific founda- tion for the specific techniques used by the FBI to determine whether these techniques met appropriate standards for scientific reliability and for use in forensic validation, and whether the FBI reached appropriate scientific con- clusions from its use of these techniques. In instances where novel scientific methods were developed for purposes of the FBI investigation itself, the com- mittee will pay particular attention to whether these methods were appropriately validated. The committee will review and assess scientific evidence (studies, results, analyses, reports) considered in connection with the 2001 Bacillus anthracis mailings. In assessing this body of information, the committee will limit its inquiry to the scientific approaches, methodologies, and analytical techniques used during the investigation of the 2001 B. anthracis mailings. The areas of scientific evidence to be studied by the committee include, but may not be limited to: 1. genetic studies that led to the identification of potential sources of B. anthracis recovered from the letters; 2. analyses of four genetic mutations that were found in evidence and that are unique to a subset of Ames strain cultures collected during the investigation; 3. chemical and dating studies that examined how, where, and when the spores may have been grown and what, if any, additional treatments they were subjected to; 4. studies of the recovery of spores and bacterial DNA from samples col- lected and tested during the investigation; and 5. the role that cross contamination might have played in the evidence picture. The committee will necessarily consider the facts and data surrounding the investigation of the 2001 Bacillus anthracis mailings, the reliability of the prin- ciples and methods used by the FBI, and whether the principles and methods were applied appropriately to the facts. The committee will not, however, under- take an assessment of the probative value of the scientific evidence in any specific component of the investigation, prosecution, or civil litigation and will offer no view on the guilt or innocence of any person(s) in connection with the 2001 B. anthracis mailings, or any other B. anthracis incidents.

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3 SUMMARY in the investigation formed the basis of a rapidly developing but still nascent scientific field, called “microbial forensics,” involving a series of laboratory tests used to determine the genetic identity of a microbial agent used for nefarious purposes. The development and application of microbial forensics would become an essential part of the scientific investigation in the hands of FBI investigators, who would combine it with physicochemical analyses to narrow their search for the possible origin of the anthrax used in the attacks. Key scientific questions focused on how, where, and when the material might have been produced; whether the material in all the evidence collected was identical; whether the material had been produced in such a manner as to make it more easily dispersible; whether it had any distinguishing physical or chemical properties of value in comparison studies; and whether its biological characteristics could provide leads to its origins. The committee carried out its work mindful of the need to identify lessons that could be learned for future investigations in which science might play an important role. Under the terms of the NRC contract with the FBI, the committee was initially provided with two boxes containing approximately 9,000 pages of materials regarding the scientific investigations undertaken by the FBI and by external experts contracted by the FBI during the investigation. At the end of the study an additional 641 pages were provided to the committee. Throughout the NRC study process, these FBI-provided materials were covered by FOIA Exemption 7, law enforcement, and were not publicly available. With the release of this report, as specified in the contract, these documents have been deposited in the NRC Public Access File.1 In addition to these materials the FBI briefed the committee on several occasions. Some of these briefings were done in open session, while others were conducted in closed sessions covered by FOIA Exemption 7. The committee also heard from a number of other experts. In conducting its review, the committee was mindful that, while its focus was on the science involved in the case, the FBI did not ask the committee to review all of the science that was used in the investigation. For example, the committee was not charged to consider or evaluate any of the traditional forensic science methods and techniques used in criminal investigations (e.g., hair, fiber, fingerprint, or handwriting analysis) (NRC, 2009a) nor did it consider any of the psychological or behavioral sciences, such as linguistics, used by the FBI in its investigation. As such, this report and the committee’s review and evaluation focused on the application of biological, physical, and chemical sciences to evi- dentiary materials from the letters, to the collection and analysis of environmental samples, to the analysis of the flask designated RMR-1029 (a flask containing 1 The public can gain access to these materials by contacting the NRC Public Access Records Office.

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4 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS anthrax spores that had been housed and maintained in a U.S. Army Medical Research Institute of Infectious Diseases [USAMRIID] laboratory at Fort Detrick since 1997), and to the collection and analysis of the B. anthracis samples from domestic and international laboratories and stored in the FBI Repository (FBIR). During the course of the NRC committee’s deliberations, the DOJ announced on February 19, 2010, that it was closing the case based on its conclusion that Dr. Bruce Ivins, a scientist at USAMRIID, had alone perpe - trated the attacks. Dr. Ivins died on July 29, 2008, after taking an overdose of over-the-counter medications. FBI SCIENTIFIC CONCLUSIONS AND COMMITTEE FINDINGS The FBI drew a number of conclusions from its scientific investigation, which are summarized in Table S-1 at the end of the Summary. The committee found it challenging, however, to identify a definitive set of scientific conclu - sions drawn by the FBI investigators because they did not provide them in written form and because the conclusions provided publicly by DOJ in its brief- ings and Investigative Summary2 varied from those provided by FBI officials in presentations to the committee. For the purposes of this report, the committee’s analyses are based on the scientific conclusions provided by the FBI to the committee on September 24, 2009 (left-most column of Table S-1) and those issued publicly by DOJ on February 19, 2010, when it closed the case (USDOJ, 2010) (column second from the left in Table S-1). The committee was not in a position to offer a judgment about the importance and strength of the evidence from the scientific investigation relative to the importance and strength of the evidence from the criminal investigation because it was not charged with (and lacked the expertise for) reviewing the latter. A summary of the committee’s findings with regard to the scientific investi - gation and the scientific conclusions that were drawn from it by the FBI is pro - vided below. The numbered statements below in bold that are labeled with an “S” (e.g., S.1) summarize subsets of the committee’s findings and are intended to organize the findings and help guide the reader through them. SUMMARY OF COMMITTEE FINDINGS It is not possible to reach a definitive conclusion about the origins of the B. anthracis in the mailings based on the available scientific evidence alone. S.1 The B. anthracis in the letters was the Ames strain and was not genetically engineered. 2 United States Department of Justice. Amerithrax Investigative Summary. February 19, 2010. Available at: www.justice.gov/amerithrax/docs/amx-investigative-summary.pdf.

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5 SUMMARY As background, the Ames strain of B. anthracis was originally isolated from a dead cow in Texas in 1981 and shipped to USAMRIID in Frederick, Maryland. Over time it was shared with research and development laboratories around the world. • The dominant organism found in the letters was correctly and efficiently identified as the Ames strain of B. anthracis. The science performed on behalf of the FBI for the purpose of Bacillus species and B. anthracis strain identifica- tion was appropriate, properly executed, and reflected the contemporary state of the art. (Finding 5.13) • The initial assessment of whether the B. anthracis Ames strain in the letters had undergone deliberate genetic engineering or modification was timely and appropriate, though necessarily incomplete. The genome sequences of the letter isolates that became available later in the investigation strongly sup - ported the FBI’s conclusion that the attack materials had not been genetically engineered. (Finding 5.2) S.2 Multiple distinct colony morphological types, or morphotypes, of B. anthracis Ames were present in the letters. Molecular assays of specific genetic sequences associated with these morphotypes provided an approach to determining rela- tionships among evidentiary samples. As background, when bacteria are grown on agar plates, the descendants of single cells produce colonies. Variation in the appearance, or morphology, of these colonies (i.e., multiple morphotypes) can indicate the presence of different species or strains in the source material, each with a distinct genetic signature, or genotype. • Multiple colony morphotypes of B. anthracis Ames were present in the material in each of the three letters that were examined (New York Post, Leahy, and Daschle), and each of the phenotypic morphotypes was found to represent one or more distinct genotypes. (Finding 5.4) • Specific molecular assays were developed for some of the B. anthracis Ames genotypes (those designated A1, A3, D, and E) found in the letters. These assays provided a useful approach for assessing possible relationships among the populations of B. anthracis spores in the letters and in samples that were subsequently collected for the FBIR (see also Chapter 6). However, more could have been done to determine the performance characteristics of these assays. In addition, the assays did not measure the relative abundance of the variant morphotype mutations, which might have been valuable and could be important in future investigations. (Finding 5.5) 3 The first number in the findings corresponds to the chapter in which they are presented.

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6 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS • The development and validation of the variant morphotype mutation assays took a long time and slowed the investigation. The committee recognizes that the genomic science used to analyze the forensic markers identified in the colony morphotypes was a large-scale endeavor and required the application of emerging science and technology. Although the committee lauds and supports the effort dedicated to the development of well-validated assays and proce - dures, looking toward the future, these processes need to be more efficient. (Finding 5.6) • A distinct Bacillus species, B. subtilis, was a minor constituent of the New York Post and Brokaw (New York) letters, and the strain found in these two letters was probably the same. B. subtilis was not present in the Daschle and Leahy letters. The FBI investigated this constituent of the New York letters and concluded, and the committee concurs, that the B. subtilis contaminant did not provide useful forensic information. While this contaminant did not provide useful forensic information in this case, the committee recognizes that such biological contaminants could prove to be of forensic value in future cases and should be investigated to their fullest. (Finding 5.3) S.3 The FBI created a repository of Ames strain B. anthracis samples and performed experiments to determine relationships among the letter materials and the repository samples. The scientific link between the letter material and flask number RMR-1029 is not as conclusive as stated in the DOJ Investiga - tive Summary. • The FBI appropriately decided to establish a repository of samples of the Ames strain of B. anthracis then held in various laboratories around the world. The repository samples would be compared with the material found in the letters to determine whether they might be the source of the letter mate - rials. However, for a variety of reasons, the repository was not optimal. For example, the instructions provided in the subpoena issued to laboratories for preparing samples (i.e., the “subpoena protocol”) were not precise enough to ensure that the laboratories would follow a consistent procedure for produc - ing samples that would be most suitable for later comparisons. Such problems with the repository required additional investigation and limit the strength of the conclusions that can be drawn from comparisons of these samples and the letter material. (Finding 6.1) • The FBI faced a difficult challenge in assembling and annotating the repository of B. anthracis Ames samples collected for genetic analysis. (Find- ing 6.9) • The results of the genetic analyses of the repository samples were con- sistent with the finding that the spores in the attack letters were derived from RMR-1029, but the analyses did not definitively demonstrate such a relation - ship. (Finding 6.2)

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7 SUMMARY • Some of the mutations identified in the spores of the attack letters and detected in RMR-1029 might have arisen by parallel evolution rather than by derivation from RMR-1029. This possible explanation of genetic similarity between spores in the letters and in RMR-1029 was not rigorously explored during the course of the investigation, further complicating the interpretation of the apparent association between the B. anthracis genotypes discovered in the attack letters and those found in RMR-1029. (Finding 6.3) • The flask designated RMR-1029 was not the immediate, most proxi- mate source of the letter material. If the letter material did in fact derive from RMR-1029, then one or more separate growth steps, using seed material from RMR-1029 followed by purification, would have been necessary. Further- more, the evidentiary material in the New York letters had physical properties that were distinct from those of the material in the Washington, D.C. letters. (Finding 4.6) • The genetic evidence that a disputed sample submitted by the suspect came from a source other than RMR-1029 was weaker than stated in the Department of Justice Amerithrax Investigative Summary. (Finding 6.4) • The scientific data generated by and on behalf of the FBI provided leads as to a possible source of the anthrax spores found in the attack letters, but these data alone did not rule out other sources. (Finding 6.5) • Biological material from all four letters should have been examined to determine whether they each contained all four genetic markers used in screen- ing the repository samples. (Finding 6.7) S.4 Silicon was present in the letter powders but there was no evidence of intentional addition of silicon-based dispersants. While any deliberate mailing of letters containing anthrax spores might be considered a form of spore “weaponization,” this term has been more com - monly used to describe preparations with enhanced properties of dispersion and aerosolization. It is commonly believed that deliberate efforts to make a powder more dispersible through the use of additives would suggest a more sophisticated level of preparation expertise. Thus, the presence of dispersants, such as nanoparticulate silica or bentonite, was an important feature in consid - ering whether or not the letters contained “weaponized” anthrax spores. • Although significant amounts of silicon were found in the powders from the New York Post, Daschle, and Leahy letters, no silicon was detected on the outside surface of spores where a dispersant would reside. Instead, significant amounts of silicon were detected within the spore coat of some samples. The bulk silicon content in the Leahy letter matched the silicon content per spore measured by different techniques. For the New York Post letter, however, there was a substantial difference between the amount of silicon measured in bulk

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8 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS and that measured in individual spores. No compelling explanation for this difference was provided to the committee. (Finding 4.3) • Surrogate preparations of B.anthracis did reproduce physical character- istics (purity, spore concentration, dispersibility) of the letter samples, but did not reproduce the large amount of silicon found in the coats of letter sample spores. (Finding 4.4) S.5 It is difficult to draw conclusions about the amount of time needed to prepare the spore material or the skill set required of the perpetrator. • The committee finds no scientific basis on which to accurately estimate the amount of time or the specific skill set needed to prepare the spore mate - rial contained in the letters. The time might vary from as little as 2 to 3 days to as much as several months. Given uncertainty about the methods used for preparation of the spore material, the committee could reach no significant conclusions regarding the skill set of the perpetrator. (Finding 4.1) S.6 Physicochemical and radiological experiments were properly conducted to evaluate the samples for potential signatures connecting them to a source but proved to be of limited forensic value. • The physicochemical methods used primarily by outside contractors early in the investigation were conducted properly. (Finding 4.2) • Radiocarbon dating of the Leahy letter material indicates that it was produced after 1998. (Finding 4.5) S.7 There was inconsistent evidence of B. anthracis Ames DNA in environ- mental samples that were collected from an overseas site. (Finding 3.4) • At the end of this study, the committee was provided limited infor- mation for the first time about the analysis of environmental samples for B. anthracis Ames from an undisclosed overseas site at which a terrorist group’s anthrax program was allegedly located. This site was investigated by the FBI and other federal partners as part of the anthrax letters investiga - tion. The information indicates that there was inconsistent evidence of Ames strain DNA in some of these samples, but no culturable B. anthracis. The committee believes that the complete set of data and conclusions concerning these samples, including all relevant classified documents, deserves a more thorough scientific review. S.8 There are other tools, methods, and approaches available today for a sci - entific investigation like this one.

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9 SUMMARY • Investigators used reasonable approaches in the early phase of the inves- tigation to collect clinical and environmental samples and to apply traditional microbiological methods to their analyses. Yet during subsequent years, the investigators did not fully exploit molecular methods to identify and character- ize B. anthracis directly in crime scene environmental samples (without culti- vation). Molecular methods offer greater sensitivity and breadth of microbial detection and more precise identification of microbial species and strains than do culture-based methods. (Finding 3.3) • Point mutations should have been used in the screening of evidentiary samples. (Finding 6.6) • New scientific tools, methods, and insight relevant to this investiga- tion became available during its later years. An important example is high- throughput, “next-generation” DNA sequencing. The application of these tools, methods, and insight might clarify (strengthen or weaken) the inference of an association between RMR-1029 and the spores in the attack letters. Such approaches will be important for use in future cases. (Finding 6.8) • The evidentiary material from this case is and will be immensely valu- able, especially in the event of future work on either this case or other cases involving biological terrorism or warfare. It is critically important to continue to preserve all remaining evidentiary material and samples collected during the course of this (the anthrax letters investigation) and future investigations, including overseas environmental samples, for possible additional studies. (Finding 6.10) S.9 Organizational structure and oversight are critical aspects of a scientific investigation. The FBI generated an organizational structure to accommodate the complexity of this case and received the advice of prominent experts. • Over the course of the investigation, the FBI found and engaged highly qualified experts in some areas. It benefited from the unprecedented guidance of a high-level group of agency directors and leading scientists. The members of this group had top secret national security clearances, met regularly over several years in a secure facility, and dealt with classified materials. The NRC committee authoring this report, in keeping with a commitment to make this report available to the public, did not see these materials. (Finding 3.1) • A clear organizational structure and process to oversee the entire scien- tific investigation was not in place in 2001. In 2003, the FBI created a new orga- nizational unit (the Chemical, Biological, Radiological, and Nuclear [CBRN] Sciences Unit, sometimes referred to as the Chemical Biological Science Unit, or CBSU) devoted to the investigation of chemical, biological, radiological, and nuclear attacks. The formation of this new unit with clearer lines of authority is commendable. (Finding 3.2)

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10 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS • As was done in the anthrax investigation, at the outset of any future investigation the responsible agencies will be aided by a scientific plan and deci- sion tree that takes into account the breadth of available physical and chemical analytical methods. The plan will also need to allow for possible modification of existing methods and for the development and validation of new methods. (Finding 3.5) LOOKING TO THE FUTURE While much of the committee’s effort was focused on a review of the sci - ence performed in support of the investigation of the 2001 B. anthracis mail- ings, an equally important goal has been to help ensure that future scientific investigations of biological attacks are conducted in the most relevant, rigor- ous, and thoughtful manner possible. Although the events of 2001 were tragic, they could have been more catastrophic. In the future, among many other requirements, it will be important to ensure more timely results, more efficient environmental analysis, access to globally representative strain collections, and a robust capability for characterizing less well studied or less easily cultivated biological agents. Officials may also need to manage expectations among the general public, policymakers, and the scientific community about the conclu - sions that can realistically be expected from the use of microbial forensics. S.10 A review should be conducted of the classified materials that are relevant to the FBI’s investigation of the 2001 Bacillus anthracis mailings, including all of the data and material pertaining to the overseas environmental sample col - lections. (Recommendation 3.1) The committee did not receive nor review classified material. In November 2010 discussions with FBI and DOJ leadership regarding this report, we were made aware of additional information that would require review of classified material. Due to the lateness of this revelation, the importance placed on issu - ing a timely report, and the agreement between the NRC and the FBI that all materials we considered be publicly available, the committee did not undertake this additional review of classified material. S.11 The goals of forensic science and realistic expectations and limitations regarding its use in the investigation of a biological attack must be com- municated to the public and policymakers with as much clarity and detail as possible before, during, and after the investigation. (Recommendation 3.2)

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11 SUMMARY TABLE S-1 FBI and DOJ Conclusions and Committee Comments Relevant report finding/ FBI conclusions DOJ conclusions Committee comment section “Spores of such high The committee finds Finding 4.1 concentration and purity no scientific basis on indicate that they were which to accurately derived from high quality estimate the amount of spore preparations. time or the specific skill Spores of this quality are set needed to prepare often used in biodefense the spore material research, including vaccine contained in the letters. development. It is important The time might vary to have highly concentrated from as little as 2 to spores to challenge most 3 days to as much as effectively the vaccine being several months. Given tested. Similarly, highly uncertainty about purified spores are necessary the methods used for to prevent obstruction of preparation of the spore the machinery used in those material, the committee experiments. These findings could reach no meant that the anthrax significant conclusions mailer must have possessed regarding the skill set of significant technical skill” the perpetrator. (USDOJ, 2010, p. 14). continued

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14 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS TABLE S-1 Continued Relevant report finding/ FBI conclusions DOJ conclusions Committee comment section Tests led to the “Two separate production The flask designated Finding 4.6 conclusion that batches of anthrax were RMR-1029 was not two separate used for the New York the immediate, most production and Washington, D.C., proximate source of the batches of mailings because each letter material. If the anthrax were contained differences in letter material did in used for the spore concentrations, color, fact derive from RMR- New York and contaminants, texture, 1029, then one or more Washington, growth media remnants, and separate growth steps, D.C. mailings observed debris. [But] when using seed material from because each coupled with the genetic RMR-1029 followed contained analysis, investigators were by purification, would differences able to conclude that the two have been necessary. in spore distinct batches of anthrax Furthermore, the concentrations, used in the 2001 attacks evidentiary material in color, shared a common origin” the New York letters contaminants, (USDOJ, 2010, p. 15). had physical properties texture, growth that were distinct from media remnants, those of the material in and observed the Washington, D.C. debris. When letters. coupled with the genetic analysis discussed in Section B, infra, investigators were able to conclude that the two distinct batches of anthrax used in the 2001 attacks shared a common origin (FBI, 2009). “This strain, known as The dominant organism Finding 5.1 ‘Ames,’ was isolated in found in the letters was Texas in 1981, and then correctly and efficiently shipped to USAMRIID, identified as the Ames where it was maintained strain of B. anthracis. thereafter.”(USDOJ, 2010, p. 3).

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15 SUMMARY TABLE S-1 Continued Relevant report finding/ FBI conclusions DOJ conclusions Committee comment section There was no “The spore particles... The initial assessment Finding 5.2 deliberate genetic showed no signs of genetic of whether the B. engineering of engineering” (USDOJ, 2010, anthracis Ames strain the B. anthracis p. 14). in the letters had strain (FBI, undergone deliberate 2009). genetic engineering or modification was timely and appropriate, though necessarily incomplete. The genome sequences of the letter isolates that became available later in the investigation strongly supported the FBI’s conclusion that the attack materials had not been genetically engineered. continued

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16 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS TABLE S-1 Continued Relevant report finding/ FBI conclusions DOJ conclusions Committee comment section The B. subtilis A distinct Bacillus Finding 5.3 contamination species, B. subtilis, was found in the a minor constituent of New York the New York Post and samples “did Brokaw (New York) not provide…a letters, and the strain productive found in these two avenue in terms letters was probably the of…leads for the same. B. subtilis was not investigation” present in the Daschle (FBI, 2009). and Leahy letters. The FBI investigated this constituent of the New York letters and concluded, and the committee concurs, that the B. subtilis contaminant did not provide useful forensic information. While this contaminant did not provide useful forensic information in this case, the committee recognizes that such biological contaminants could prove to be of forensic value in future cases and should be investigated to their fullest. There were Multiple colony Finding 5.4 mainly wild- morphotypes of type B. anthracis B. anthracis Ames Ames strain, were present in the but there were material in each of significant the three letters that numbers of were examined (New phenotypic York Post, Leahy, and variants or Daschle), and each substrains (FBI, of the phenotypic 2009). morphotypes was found to represent one or more distinct genotypes.

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17 SUMMARY TABLE S-1 Continued Relevant report finding/ FBI conclusions DOJ conclusions Committee comment section These phenotypic “Genetic analysis of Specific molecular Finding 5.5 variants could morphological variants assays were developed be detected by identified mutations which for some of the a combination were later exploited to B. anthracis Ames of assays for develop specific assays genotypes (those four different to identify the presence designated A1, A3, insertion/deletion of identical mutations in D, and E) found in polymorphisms evidence collected during the letters. These (FBI, 2009). the investigation” (USDOJ, assays provided a 2010, pp. 24-25). useful approach for assessing possible relationships among the populations of B. anthracis spores in the letters and in samples that were subsequently collected for the FBI Repository (FBIR) (see Chapter 6). However, more could have been done to determine the performance characteristics of these assays. In addition, the assays did not measure the relative abundance of the variant morphotype mutations, which might have been valuable and could be important in future investigations. There was inconsistent Finding 3.4 evidence of B. anthracis Ames DNA in environmental samples that were collected from an overseas site. continued

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18 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS TABLE S-1 Continued Relevant report finding/ FBI conclusions DOJ conclusions Committee comment section “…fifteen domestic The FBI repository Section 6.2 laboratories and three was developed from foreign laboratories were an intensive effort to identified as repositories of identify laboratories Ames strain anthrax at the having access to the time of the letter attacks.” Ames strain; however, (USDOJ, 2010, p. 17). we cannot conclude that this approach identified “…the collection of Ames every laboratory or isolates from laboratories was a comprehensive both from the United representation. States and abroad that constitute the FBIR are a comprehensive representation of the Ames strain (USDOJ, 2010, p. 28). “A total of 1, 070 samples were ultimately submitted [to the FBIR], which represents a sample from every Ames culture at every laboratory identified by the FBI as having Ames strain” (USDOJ, 2010, p. 24).

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19 SUMMARY TABLE S-1 Continued Relevant report finding/ FBI conclusions DOJ conclusions Committee comment section “FBI collaborated with The FBI appropriately Finding 6.1 various experts…to provide a decided to establish a clear and thorough protocol repository of samples for the preparation of the of the Ames strain of repository submissions” B. anthracis then held (USDOJ, 2010, p. 77). in various laboratories around the world. The repository samples would be compared with the material found in the letters to determine whether they might be the source of the letter materials. However, for a variety of reasons, the repository was not optimal. For example, the instructions provided in the subpoena issued to laboratories for preparing samples (i.e., the “subpoena protocol”) were not precise enough to ensure that the laboratories would follow a consistent procedure for producing samples that would be most suitable for later comparisons. Such problems with the repository required additional investigation and limit the strength of the conclusions that can be drawn from comparisons of these samples and the letter material. continued

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20 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS TABLE S-1 Continued Relevant report finding/ FBI conclusions DOJ conclusions Committee comment section “In 2007, after several years The results of the Finding 6.2 of scientific developmens genetic analyses of the and advanced genetic testing repository samples coordinated by the FBI were consistent with Laboratory, the Task Force the finding that the determined that the spores in spores in the attack the letters were derived from letters were derived a single spore-batch of Ames from RMR-1029, but strain anthrax called “RMR- the analyses did not 1029” (USDOJ, 2010, p. 5.) definitively demonstrate “Later in the investigation, such a relationship. The when scientific breakthroughs scientific data alone do led investigators to conclude not support the strength that RMR-1029 was the of the government’s parent material to the repeated assertions that anthrax powder used in that “RMR-1029 was the mailings,..” (USDOJ, conclusively identified 2010, p. 6). “After a time- as the parent material consuming process, the to the anthrax powder scientific analysis coordinated used in the mailings” by the FBI Laboratory (USDOJ, 2010, p. 20), determined that RMR-1029, nor the role suggested a spore-batch created and for the scientific data maintained at USAMRIID in arriving at their by Dr. Ivins, was the parent conclusions, “the material for the anthrax used scientific analysis in the mailings.” (USDOJ, coordinated by the FBI 2010, p. 8). ..genetic analysis Laboratory determined led to the conclusion that that RMR-1029, a RMR-1029, located at spore-batch created USAMRIID, was the parent and maintained at material mailed so the mailed USAMRIID by Dr. spores,..: (USDOJ, 2010, Ivins, was the parent p. 16 material for the anthrax “…based on advanced used in the mailings” (USDOJ, 2010, p. 8).b genetic testing combined with rigorous investigation, the FBI concluded that RMR- 1029 is the parent material of the evidentiary anthrax spore powder, i.e., the evidentiary material came from a derivative growth of RMR- 1029” (USDOJ, 2010, p. 28).

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21 SUMMARY TABLE S-1 Continued Relevant report finding/ FBI conclusions DOJ conclusions Committee comment section “If Dr. Ivins prepared his The genetic evidence Finding 6.4 submission to the repository that a disputed sample in accordance with the submitted by the protocol, that submission suspect came from could not miss all four of a source other than the morphological variants RMR-1029 was weaker present in RMR-1029” than stated in the (USDOJ, 2010, p. 79). Department of Justice Amerithrax Investigative Summary. “…the only complete genetic The scientific data Finding 6.5 match to the evidence comes generated by and on from RMR-1029 and its behalf of the FBI offspring” (USDOJ, 2010, provided leads as to a p. 29). possible source of the anthrax spores found in the attack letters, but these data alone did not rule out other sources. continued

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22 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS TABLE S-1 Continued Relevant report finding/ FBI conclusions DOJ conclusions Committee comment section Out of the The FBI and contract Finding 6.1 1,059 viable scientists appropriately discussion samples from recognized that the various stocks mutations in the letter collected in the isolates provided FBIR during the information that might investigation, help identify the source 8 contained of the B. anthracis all 4 of the used in the attacks, polymorphisms; developed appropriate 2 contained assays for four of 3 of the 4 these mutations, and polymorphisms; created and screened and a few a repository of Ames contained 1 or 2 strain samples. Based of the mutations. on the results of that (FBI, 2009). screening, FBI scientists appropriately concluded that the majority of repository samples contained none of the four mutations, although 50 of the samples contained one of the four mutations and 10 samples had three or all four mutations (the numbers with one or more mutations are higher if one includes samples that were excluded in the FBI’s Statistical Analysis Report). However, features of the repository, including unknown provenance, possibly multiple samples from the same flask, the history of sharing and mixing of stocks presented investigative challenges.

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23 SUMMARY TABLE S-1 Continued aNote that this was the final conclusion of the scientific investigators. An initial finding by the Armed Forces Institute of Pathology (AFIP) found, upon gross examination, that the spores exhib- ited a silicon signal and sometimes exhibited an oxygen signal. Subsequent studies conducted by Sandia National Laboratories (as described in Chapter 4 of this report) determined that the silicon was localized to the spore coat within the exosporium—that is, it was incorporated into the cell as a natural part of the cell formation process. The USAMRIID scientist who first reviewed the AFIP results and made statements regarding the presence of silicon and possible weaponization retracted those earlier statements. bSee for example, “As noted above, based on advanced genetic testing combined with rigorous investigation, the FBI concluded that RMR-1029 is the parent material of the evidentiary anthrax spore powder, i.e., the evidentiary material came from a derivative growth of RMR-1029.” (p. 28) “As described in detail above, over time, genetic analysis determined that one of Dr. Ivins’s Ames cultures, RMR-1029—the purest and most concentrated batch of Ames spores known to exist—was the parent to the evidentiary material used in the anthrax mailings.” (p.79)

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