Review of the Scientific Approaches Used During the FBI's Investigation of the 2001 Anthrax Letters (2011) / Chapter Skim
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6 Comparison of the Material in the Letters with Samples in the FBI Repository
6 Comparison of the Material in the Letters with Samples in the FBI Repository
Pages 125-152
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From page 125... ...
Particular attention is given to the limitations of applying formal statistical methods to these results and of trying to quan tify the strength of the relationship between the spores in the attack letters and those in RMR-1029. The committee then discusses evidence bearing on a disputed sample submitted by the suspect in this case as well as follow-up experiments performed by the FBI to determine whether that sample had come from its stated source, RMR-1029 (see Amerithrax Investigative Summary, 125
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From page 126... ...
anthracis, and FBI investigators created their own list based on CDC select agent transfer records documenting every transfer of anthrax between 1997 and 2001 as well as anthrax inventory records
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From page 127... ...
Source: FBI Documents, Preparing and Shipping TSA Slants for B Anthracis Ames.
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From page 128... ...
The FBI described their approach to assessing Ames strains internationally in the following way: Complementing this initiative was a separate intensive effort to assess whether a foreign government or a terrorist organization may have gained access to the Ames strain and perpetrated the attacks. An exhaus tive initiative, one that continued until the closing of the case, addressed this concern.
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From page 129... ...
Given uncertainties in the extent of the entire population of Ames stocks worldwide (highlighted by concern about the possibility of clandestine stocks held by terrorist organizations -- see section 3.4.3) , the lack of specificity in the subpoena protocol, the uncertain ties in compliance with the subpoena protocol, the incomplete information on transfers of Ames-derived stocks between laboratories, and the possibility that some stocks were produced but later destroyed, the repository was unlikely to have been comprehensive.2 DOJ states in its Summary, "The collection of Ames isolates from laboratories both from the United States and abroad that consti tute the FBIR are a comprehensive representation of the Ames strain" (USDOJ, 2010, p.
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6.3 USE OF THE GENETIC ASSAYS TO TEST FOR THE FOUR GENOTYPES Either during or after the development of the genetic assays, a decision was made that they should be performed on samples of B anthracis Ames only after the cells in the sample were cultivated once again (in addition to the prior cul tivation step by the recipients of the subpoena protocol before submitting the samples)
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From page 131... ...
The production history of the RMR-1029 spore population provides a probable explanation for the presence of subpopulations of genetic variants, including variants that could give rise to visually distinctive colonies (morphotypes)
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From page 132... ...
The purpose of these assays was to search for the presence of the genetic mutations -- A1, A3, D, and E -- among the FBIR samples to determine whether any of the samples had a genetic profile that matched that of the evidentiary material and could be a possible source of the material used in the letters. It is important to reiterate here that the existence in laboratories of the Ames strain of B
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From page 133... ...
in the Statistical Analysis Report. The FBI did not seek formal statistical expertise until it had completed the genotype assays of the repository samples.
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From page 134... ...
An additional 112 samples were omitted from further investigation because the results with these samples were recorded as "inconclusive," "variant," "no growth," or "no DNA" for one or more of the four genotypes. Tables 6-2 and 6-3 summarize the data from the Statistical Analysis Report in two ways, in each case using only the 947 samples that produced definitive results.
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(c) Relationships among samples Given the existence of historical relationships among the 947 repository samples, the Statistical Analysis Report described two methods to assess these relationships.
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of the other nine laboratories or institutions that submitted samples that were positive for one or more of the four genotypes. Based on these two methods, the Statistical Analysis Report concluded that there were associations between the eight samples testing posi tive for four genotypes and the other samples that were positive for one or more of the genotypes.
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From page 137... ...
The Statistical Analysis Report includes a formal statistical test to assess the significance of the differences between these "observed frequencies" and the "expected frequencies." However, the inference from this test is not valid because, as noted above, the 947 samples were not independent. The committee identified two potential sources of dependence among repository samples in the results of the four genotype assays.
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From page 138... ...
While assay sensitivity and specificity were assessed by the assay development contractors in an idealized and artificial setting during the development of the assays, a more meaningful assessment in the context of the testing of actual repository samples would require replication of tests using FBIR samples, or the creation of multiple types of simulated stock samples. To assess the sensitivity of the assays, dilution experiments were conducted on two samples, RMR-1029 and "SPS.266 Tube#5," using three replicates at each of 10 dilution levels.
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Table 6-5 provides the results from these dilution experiments summarized in the Statistical Analysis Report. The lack of agreement between MRI and IITRI assays for the D genotype has already been noted (Table 6-1)
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. The lack of replication in the assays of the FBIR samples makes it impossible to quantify the strength of any finding relating to the presence or absence of genotypes in the repository samples since some absences may be false negatives.
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From page 141... ...
and also focused on secondary analyses performed in order to reconcile certain discrepancies in the multiple samples that were submitted by Ivins and purported to come from flask RMR-1029. FBI investigators had observed that the second RMR-1029 sample submitted by Ivins in April 2002 did not score positive for any of the four genotypes discovered in the attack letters and in RMR-1029 as well as in samples derived from RMR-1029 that were submitted by other scientists.
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From page 142... ...
None of the 30 samples scored negative for all four mutations. Given these assay results of the 30 replicates, what is the probability that analysis on an additional sample, taken from RMR-1029 in the same manner, would yield negative results for all four genotypes by chance alone?
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143 COMPARISON OF THE MATERIAL WITH SAMPLES IN THE FBI REPOSITORY TABLE 6-6 Results Obtained by Resampling from Flask RMR-1029 A1 A3 D IITRI D MRI E Sample 1 + + + + + Sample 2 + + + + + Sample 3 + + + + + Sample 4 + + + + + Sample 5 + + + + + Sample 6 + + + + + Sample 7 + + Negative + + Sample 8 + + + + + Sample 9 + + + + + Sample 10 Inconclusive + Inconclusive Inconclusive + Sample 11 + + + + + Sample 12 Inconclusive + Inconclusive Negative + Sample 13 Inconclusive + Negative Inconclusive + Sample 14 Negative + + + + Sample 15 Negative + + + + Sample 16 + + + + + Sample 17 + + + + + Sample 18 Negative + + + + Sample 19 + + + + + Sample 20 Negative Inconclusive Negative Negative + Sample 21 + + + + + Sample 22 Negative + + + + Sample 23 + + + + + Sample 24 Negative + Negative Negative + Sample 25 Inconclusive + + + + Sample 26 + + + + + Sample 27 Negative + + + + Sample 28 + + + + + Sample 29 Negative + + + + Sample 30 Negative + Inconclusive Negative + NOTES: += positive ; IITRI = IIT Research Institute; MRI = Midwest Research Institute SOURCE: FBI Documents, B2M10D2.
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From page 144... ...
anthracis used in the attacks, developed appropriate assays for four of these mutations, and created and screened a repository of Ames strain samples. Based on the results of that 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 report)
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From page 145... ...
Ivins. Finding 6.2: The results of the genetic analyses of the repository samples were consistent with the finding that the spores in the attack letters were derived from RMR-1029, but the analyses did not definitively demonstrate such a relationship.
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anthracis used in the three attack letters that were tested; however, there are several important caveats. As discussed above, the nature of the repository collection, including the incompletely documented history of sharing and mixing of Ames strain stocks and the ambiguity in the subpoena protocol, makes it difficult to quantify the strength of the evidence linking RMR-1029 to the letters, because of the complex and ill-defined nature of the reference population.
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From page 147... ...
Finding 6.4: 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. The committee reexamined the data that the FBI obtained following the discovery that one of the samples submitted by Bruce Ivins, which was sup posed to have been taken from RMR-1029, did not test positive for any of the four assayed mutations (A1, A3, D, and E)
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From page 148... ...
The FBI observed morphological variants in the B anthracis isolated from the attack letters and used those variants to identify mutations that were then used as genetic markers during the systematic screening of the repository samples.
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From page 149... ...
Since 2001, important technological advances have occurred that would allow a more thorough and systematic analysis of genetic similarities and dif ferences between key evidentiary samples, repository samples, and appropri ate controls. When the investigation began, bacterial genomes were typically sequenced at an average coverage of fewer than 10 reads per nucleotide (see, for example, Read et al., 2002)
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From page 150... ...
Given the limitations of the existing repository, it is uncertain whether this further scientific investi gation would have identified an altogether different source of the B anthracis attack spores, but it could have provided additional information on the process used to generate the material in the attack letters.
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From page 151... ...
Finding 6.10: The evidentiary material from this case is, and will be, immensely valuable, 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)
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