Published Date: 2011-02-16 15:00:05
Subject: PRO/AH> Anthrax, human, 2001 - USA: Nat'l Research Council rep.
Archive Number: 20110216.0511
ANTHRAX, HUMAN, 2001 - USA: NATIONAL RESEARCH COUNCIL REPORT
A ProMED-mail post
ProMED-mail is a program of the
International Society for Infectious Diseases
Date: Tue 15 Feb 2011
Source: National Research Council (NRC) of the National Academies,
The National Academies Press [edited]
[Copies may be purchased from that site. There is also the recording
there of the 65-minute 15 Feb 2011 press meeting. - Mod.MHJ]
Review of the scientific approaches used during the FBI's [Federal
Bureau of Investigation] investigation of the 2001 anthrax letters
Summary of Committee findings
Major finding: it is not possible to reach a definitive conclusion
about the origins of the _Bacillus 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.
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 relationships among evidentiary samples.
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 [Department of Justice] Investigative
S.4: Silicon was present in the letter powders but there was no
evidence of intentional addition of silicon-based dispersants.
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
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.
S.7: There was inconsistent evidence of _B. anthracis_ Ames DNA in
environmental samples that were collected from an overseas site.
S.8: There are other tools, methods, and approaches available today
for a scientific investigation like this one.
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.
S.10: A review should be conducted of the classified materials that
are relevant to the FBI's investigation of the 2001 _B. anthracis_
mailings, including all of the data and material pertaining to the
overseas environmental sample collections. (Recommendation 3.1)
S.11: The goals of forensic science and realistic expectations and
limitations regarding its use in the investigation of a biological
attack must be communicated to the public and policymakers with as
much clarity and detail as possible before, during, and after the
investigation. (Recommendation 3.2)
Findings and recommendations
Finding 3.1: 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 3.2: A clear organizational structure and process to oversee
the entire scientific investigation was not in place in 2001. In 2003,
the FBI created a new organizational 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 3.3: Investigators used reasonable approaches in the early
phase of the investigation 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 characterize _B. anthracis_
directly in crime scene environmental samples (without cultivation).
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 3.4: There was inconsistent evidence of _B. anthracis_ Ames
DNA in environmental samples that were collected from an overseas
Finding 3.5: 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 decision 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 (see
Chapter 4, Section 12).
***Recommendation 3.1: A review should be conducted of the classified
materials that are relevant to the FBI's investigation of the 2001 _B.
anthracis_ mailings, including all of the data and material pertaining
to the overseas environmental sample collections.
***Recommendation 3.2: The goals of forensic science and realistic
expectations and limitations regarding its use in the investigation of
a biological attack must be communicated to the public and
policymakers with as much clarity and detail as possible before,
during, and after the investigation.
Finding 4.1: 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 material 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 4.2: The physicochemical methods used primarily by outside
contractors early in the investigation were conducted properly.
Finding 4.3: 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 coats 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 and that measured in individual spores. No
compelling explanation for this difference was provided to the
Finding 4.4: Surrogate preparations of _B. anthracis_ did reproduce
physical characteristics (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. [The committee
appears to have ignored how these significantly high levels of silicon
within the spore coat were achieved ... usually ascribed to the use of
silanes ... and thus where such a technical skill could be found. -
Finding 4.5: Radiocarbon dating of the Leahy letter material
indicates that it was produced after 1998.
Finding 4.6: The flask designated RMR-1029 was not the immediate,
most proximate 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. Furthermore, the evidentiary material in
the New York letters had physical properties that were distinct from
those of the material in the Washington, DC letters.
(Specifically) SEM-EDX measurements showed no silicon in the coats of
spores taken directly from RMR-1029, whereas the majority of spores
analyzed from the New York Post, Daschle, and Leahy letter materials
contained silicon in the coat. Based on recent studies of the
mechanism of silicon incorporation, silicon could have been
incorporated in the coats of the letter spores only if spores from
RMR-1029 were subjected to one or more subsequent growth steps.
Another observation consistent with a separate growth step was the
detection of _B. subtilis_ in the New York Post and Brokaw letter
material but not in RMR-1029 (discussed in Chapter 5). The detection
of meglumine and diatrizoate in RMR-1029 but not in the Leahy and New
York Post samples also is consistent with this finding; however, it is
not conclusive because it might have been possible to rinse these
impurities away without requiring later growth. Some of these
findings, as well as others, indicate that the New York letter
materials were prepared separately from the materials in the
Washington, DC, letters. The presence of _B. subtilis_ in the New York
but not the Washington letter materials and the different physical
properties of the materials indicate that the 2 sets of letter
materials were prepared separately.
Finding 5.1: 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 identification was appropriate,
properly executed, and reflected the contemporary state of the art.
Finding 5.2: 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 supported
the FBI's conclusion that the attack materials had not been
Finding 5.3: 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 2 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. [The FBI downplayed their failure to identify a possible
source for this contaminant, a species frequently used as a stimulant
and therefore a potential institutional fingerprint as to where one
set of spores were cultured. - Mod.MHJ]
Finding 5.4: Multiple colony morphotypes of _B. anthracis_ Ames were
present in the material in each of the 3 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 5.5: 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 FBI Repository (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 5.6: 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 procedures,
looking toward the future, these processes need to be more efficient.
Finding 6.1: 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 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 (that is, 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.
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.
Finding 6.3: 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 6.4: 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
Finding 6.5: 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
Finding 6.6: Point mutations should have been used in the screening
of evidentiary samples.
Finding 6.7: Biological material from all 4 letters should have been
examined to determine whether they each contained all 4 genetic
markers used in screening the repository samples.
Finding 6.8: New scientific tools, methods, and insight relevant to
this investigation 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 6.9: The FBI faced a difficult challenge in assembling and
annotating the repository of _B. anthracis_ Ames samples collected for
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) and future investigations,
including the overseas environmental samples, for possible additional
[In a word, the scientific evidence at this time does not validate
the FBI conclusions based on their circumstantial evidence and in fact
casts a wider net. For additional comments see