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3
Scientific Investigation in a Law
Enforcement Case and Description and
Timeline of the FBI Scientific Investigation
3.1 INTRODUCTION
The discoveries that individuals had contracted anthrax and that letters
containing B. anthracis had been sent by U.S. mail launched a full-scale inves-
tigation by the U.S. Centers for Disease Control and Prevention (CDC), the
U.S. Postal Inspection Service (USPIS), and the Federal Bureau of Investiga -
tion (FBI). Numerous investigative techniques were applied throughout the
investigation, as outlined in Table 3-1. This chapter describes in brief the early
stages of the investigation, specifically the gathering of evidence, formation of
investigative teams, and decisions regarding scientific analyses that led to the
FBI findings and conclusions that are summarized and evaluated more fully
in Chapters 4 through 6.1 It also introduces the concepts of science, scientific
investigation in law enforcement, and the different views of uncertainty—and
the manner in which it is described—in science versus law.
3.2 SCIENCE AND SCIENTIFIC INVESTIGATION AS PART OF
A LAW ENFORCEMENT INVESTIGATION
In a scientific study, explanations for observable phenomena are sought
through the gathering of reliable data and the formulation of testable hypoth -
eses. Scientific observations must be reproducible and scientific hypotheses
must be refutable. Science is typically an iterative, collaborative, and open
process requiring the ability to pose hypotheses, test them, and pose new ques -
tions based on the resulting information. At times, a scientific investigation is a
divergent process, in which new results drive research in several directions only
1 Throughout this report, the committee describes its evaluation of the primary reports and data
contained in the materials provided to the committee by the FBI. In cases where the committee
was not able to review primary data, the committee’s assessment of statements or analyses of data
by others is provided.
47
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48 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
TABLE 3-1 Timeline of Scientific Events in the Anthrax Mailings
Investigation
Agency/
institution/
individual FBI
Project Final conducting the document
initiated report work Project number
10/4/01 First case of anthrax reported N/A
to CDC. The first lab
confirmation of an initial
clinical identification of B.
anthracis from a victim of
the letter attacks (Stevens)
was done at the Florida State
Laboratory in Jacksonville.
10/12/01 11/14/01 FBI and local law Collection of biological N/A
enforcement evidence: 4 envelopes,
17 clinical samples, 106
environmental samples along
mail paths (FL, DC, NJ, NY,
CT)
10/17/01 10/19/01 Battelle Memorial Microbiological analyses of B2M1D1
Institute (BMI) letter material identifies 2 B2M13D4
Bacillus species: one non-
beta-hemolytic (consistent
with B. anthracis) and one
beta-hemolytic (not further
characterized)
10/01 Beecher Environmental sampling of mail
bags
10/01 11/01 CDC Clinical isolates from stricken N/A
patients identified as B. anthracis
10/18/01 11/27/01 USAMRIID Initial characterization of letter B1M1D2
material (CFU, EM, visual
inspection)
10/01 11/26/01 Battelle Memorial SEM-EDX analysis of letter B2M13D3,
Institute material B2M13D8
11/01 Armed Forces SEM-EDX analysis of letter AFIP,
Institute of material 2001
Pathology (AFIP)
10/01 05/02 Los Alamos Material analysis for evidence B1M4
National of genetic engineering
Laboratory
(LANL)
10/01 9/02 Northern Arizona Identification of USAMRIID B1M3
University samples as Ames strain
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49
SCIENTIFIC INVESTIGATION IN A LAW ENFORCEMENT CASE
TABLE 3-1 Continued
Agency/
institution/
individual FBI
Project Final conducting the document
initiated report work Project number
Fall 2/28/02 BMI Particle size distribution B2M13D11
2001 performed on letter samples
and some surrogate samples,
but not the Dugway Proving
Ground surrogates
Fall CDC Combined epidemiological
2001 analysis of all case related to
the anthrax mailings
11/07/01 12/03/01 CDC Using phenotypic substrate B2M1D2
utilization and 16S rDNA
sequencing, Bacillus
contaminant identified as B.
subtilis
11/09/01 11/09/01 FBI SEM analysis of envelopes B1M7D16
Fall Consortium of agencies N/A
2001 (NSF, NIH, DOE, DOJ, FBI,
USDA, DOD, and Intelligence
Community) formed to advise
investigation and provide
support resources
Fall BMI Silicon incorporation into spore B2M13D7
2001 coat
Fall Spring The Institute for Completion of genome B1M5D1,
2001 2002 Genomic Research sequence for B. anthracis B1M5D3
(TIGR) Porton and “Ames 2001
Florida strain” (clinical isolate);
publication in Science, June
2002
Fall Spring USAMRIID Detection of phenotypic B1M2D12
2001 2002 variants (“morphotypes”)
among colonies derived from
letter spores
12/01 12/06 Dugway Proving Reverse engineering of spore B1M13
Ground (DPG) “powders” B1M14
continued
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50 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
TABLE 3-1 Continued
Agency/
institution/
individual FBI
Project Final conducting the document
initiated report work Project number
2/02 10/14/02 Lawrence Carbon dating by Accelerator B1M8
Livermore Mass Spectrometry
National
Laboratory
(LLNL), National
Ocean Sciences
Accelerator Mass
Spectrometry
Facility National
Ocean Sciences
Accelerator Mass
Spectrometry
Facility
(NOSAMS),
Woods Hole
Oceanographic
Institute
2/02 FBI Subpoenas of laboratories for
samples of B. anthracis Ames
2/02 10/06 Sandia National Elemental analyses (SEM-EDX) B1M7
Laboratory (SNL) of letter material, envelopes,
and DPG surrogates (final
report not dated)
2/02 10/06 SNL Silicon analyses B1M6
2/01/02 8/13/05 University of Agar analysis B1M10
Maryland (UMD)
3/07/02 2/01/06 DPG Analytical chemistry analysis of B1M13
spore powders
3/22/02 4/14/02 FBI Volatile organic compound B1M7D2
analysis in evidentiary material
Early 7/6/05 FBI ICP-OES: elemental B1M6
2002 composition of letter material, B1M7
culture media, envelope types
12/02 6/1/04 TIGR Whole genome sequencing of B1M5
Morphs A, B, C, D
8/02 2/22/04 UMD Heme analysis B1M10
8/02 8/05 Agar and heme analysis B1M10
Edgewood Chemical
Biological Center
(Army) ECBC
8/02 9/02 BMI Agar and heme analysis B1M10
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51
SCIENTIFIC INVESTIGATION IN A LAW ENFORCEMENT CASE
TABLE 3-1 Continued
Agency/
institution/
individual FBI
Project Final conducting the document
initiated report work Project number
10/02 2/04 Commonwealth Contract to develop Morph A B2M5
Biotechnologies, assays; only A1 and A3 were
Inc. (CBI) validated
3/03 5/05 University of Utah Stable isotope signatures B1M9
3/14/03 10/10/03 Applied 16S rDNA sequencing of B2M1D4
Biosystems (AB) Brokaw letter B. subtilis
contaminants
7/31/03 8/11/03 Novozymes B. subtilis contaminant B2M1D3
Biotech, Inc. compared to B. licheniformis
10/03 5/14/06 TIGR Multiple locus PCR-based B1M5D2
assay for direct comparison of
B. subtilis strains to Post B.
subtilis
10/03 6/4/05 TIGR Whole genome sequencing B1M5
of Morph E and B. subtilis
contaminant (Post and Leahy)
10/10/03 6/30/05 AB Genome sequencing of B.
subtilis “H2122” (not identified
elsewhere)
3/04 2/06 CBI Repository (1104 samples) B2M5D8
screening for A1 and A3;
second screening (300 samples)
7/07-10/07)
6/25/04 7/05 CBI Contract to develop assays for B2M6
Morphs B and D: both rejected
7/13/04 9/14/04 USAMRIID Screening of selected samples B1M2D13
of FBIR for presence of
Morphotypes
7/23/04 6/7/05 Midwest Research Contract to develop assay for B2M8
Institute (MRI) Morphs B and D; Morph B
assay rejected; Morph D assay
accepted
11/19/04 4/05 IIT Research Contract to develop assays for B2M7
Institute (IITRI) Morphs B and D; Morph B
assay rejected; Morph D assay
accepted
12/01/05 10/10/07 MRI Repository screening for Morph B2M8
D
continued
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52 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
TABLE 3-1 Continued
Agency/
institution/
individual FBI
Project Final conducting the document
initiated report work Project number
5/05 4/24/07 IITRI Repository screening for Morph B2M7
D
12/05 1/07 TIGR Contract to develop assay for B2M9
Morph E
10/06 7/07 Pacific Northwest Agar and heme analysis B1M11
National
Laboratory
(PNNL)
11/06 12/07 NBFAC Repository screening for B. B2M4D2
subtilis contaminant
2/15/07 12/04/07 CBSU (FBI) B. subtilis analysis by real-time B2M4
National PCR: screening of repository
Bioforensic and other samples
Analysis Center
(NBFAC)
6/25/07 8/25/07 TIGR Repository screening for B2M9
Morph E
8/04/07 11/30/07 National Center Genetic diversity and B2M3
for Agricultural phylogenetic analysis of B.
Utilization subtilis samples
Research
(NCAUR)
8/29/07 8/29/08 FBI Analysis of meglumine and B1M12
diatrizoate in RMR-1029, letter
material, other samples
10/05/07 10/08/08 TIGR Finalization of B. subtilis B2M2D2
genome sequence
1/09/08 6/08/08 TIGR Screening of unidentified B. B2M2D3,
subtilis isolates for presence B2M2D4
of sequence specific to Post/
Brokaw contaminant
3/27/08 9/30/08 University of Statistical analysis of FBIR B2M10
Cincinnati screening data
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53
SCIENTIFIC INVESTIGATION IN A LAW ENFORCEMENT CASE
to converge again when more information informs decisions about which direc-
tions to pursue. This divergence and convergence make a scientific investiga -
tion different from a law enforcement investigation, in which the drive toward
convergence dominates to a greater degree. In addition, the approach used
to gather, process, and analyze evidence can differ between a purely scientific
investigation and a law enforcement investigation. Both types of approaches are
necessary in a bioterrorism investigation, which also requires attention to public
health risks and safety needs (see Box 3-1).
An important feature of science is that observations are made in a manner
that is independent of the observer and on the assumption that other observers
can and would make the same observations. Science relies on validated methods
for gathering observations and making quantitative measurements systematically
and reproducibly. Standards must be set for collecting data under controlled and
well-specified conditions, assessing possible sources of error, establishing causal-
ity (and acknowledging that a relationship is only a correlation when causality
cannot be inferred and supported), and applying empirical findings to validate
or refute particular hypotheses. New scientific methods must be assessed for
their accuracy and reliability, their limitations, and the range of circumstances
under which they can be appropriately applied.
The Qualifiers of Certainty in the Biological Sciences
A key question in this study was “Based on the available data, how strong
is the apparent association between the letter evidentiary material and a par-
ticular source or sample (e.g., flask RMR-1029)?” Some of the committee’s
most important findings focus on the strength of a given association and on
the conclusions that one should draw from the available scientific data about
the nature of the association. Thus, it is important to review briefly the use of
terminology to describe the strength of an association.
Quantifying an association, as well as the degree of certainty (or uncertainty)
in that association, involves statistical methods (see Chapter 6). Common lan-
guage involves qualifiers, rather than quantifiable measures, of this association
and the degree of confidence in it, which can cause confusion among prac-
titioners from different fields that use the terms. Since the interpretation of these
qualifiers and the ways in which they are used differ across disciplines (e.g.,
statistics, science, law, common language), their use by the committee is clarified
here. In the chapters that follow, the committee uses the following four qualifiers
of association, listed in order of increasing certainty (decreasing uncertainty):
• consistent with an association
• suggest an association
• indicate an association
• demonstrate an association
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54 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
BOX 3-1
Bioterrorism Investigations
A crime scene typically is a place where the victims and perpetrators meet in time
and space. The traditional crime scene, which may have multiple locations, is the logi-
cal place to search for physical evidence leading to the identity of the perpetrator(s).
Identifying, collecting, and preserving probative evidence combined with investigative
detective work is the usual approach to a successful prosecution. Solving the crime
is the ultimate goal of the scene investigation, but there also are other reasons to
investigate the crime scene, including: 1) developing investigative leads for detec-
tives; 2) developing specific information in the form of evidence or investigative logic
to enable a successful prosecution; 3) locating, collecting, and preserving probative
physical evidence that can provide evidence of innocence or guilt; 4) developing infor-
mation and physical evidence that provides an accurate reconstruction of the events
of the crime; and 5) linking multiple crimes through the evidence collected across sites
(USDOJ, 2000; Fisher, 2005).
All crime scene investigations require the integration of multiple forensic disci-
plines through the juxtaposition of science and scene investigative skills. Scientific
criminal investigations require an amalgamation of capabilities including scene expe-
rience, attention to detail, a skeptical perspective, powers of observation, and the
application of logic (Gardner, 2005).
Although bioterrorism event scenes have elements in common with other crime
scenes, such as the identification, collection, and preservation of the forensic evi-
dence, they can also differ because of the inherent risks to investigators and to the
public. Also, unlike traditional crimes, they may not always involve a location where
the participants meet in time and space, as shown by the 2001 B. anthracis mailings
in which the dissemination of B. anthracis occurred by means of the United States
Postal Service (Jernigan et al., 2002). The perpetrator(s) presumably worked at a
distance, so the criminal investigation spanned several locations, including those of
the envelopes, post offices, and street postal boxes that might have held contaminated
envelopes, and the location(s) where the B. anthracis might have been manufactured.
Each scene required a comprehensive and coordinated investigation to find, collect,
and preserve the B. anthracis spores.
Future bioterrorism events may differ in the nature of the biological agent or
toxin and in the mode of delivery. In general, bioterrorism incidents can be expected
to be handled differently than the typical homicide scene investigation because such
events require both traditional scene management skills and the special requirements
of scenes involving bioagents. Bioterrorism investigators must consider issues such
as public safety, operational planning, sampling strategy, packaging, transport, and
storage. The immediate imperative to consider public health needs requires finding
and collecting the biological agent so that it can be identified expeditiously, as well as
defining and containing the environmental risk to those not yet exposed. The investiga-
tion and containment must be accomplished while ensuring the safety of investigators
and the public during the investigation and while remediating the scene. Sampling
strategies must combine the collection and preservation of bioagents with the collec-
tion of usual forensic evidence (Budowle, 2006). In some future scenarios, delivery
of the biological agent might occur through natural routes of biological transmission,
and thus the “crime scene” may be limited to the site at which the biological agent was
prepared or delivered.
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55
SCIENTIFIC INVESTIGATION IN A LAW ENFORCEMENT CASE
The expression “consistent with” is frequently used in this report and
conveys the weakest level of certainty (greatest amount of uncertainty). In
general, when the term “consistent with” is used, it means that an association
may or may not be present; the available data can neither rule out nor confirm
an association. The term “suggests” denotes a greater level of certainty for an
association than “consistent with,” but even here the normal use of the word
in science denotes a weaker level of certainty than is implied by the word in
everyday parlance. That is, the potential for an association is stronger, and the
evidence for the absence of an association is weaker, but both are still possible.
In contrast, the terms “indicate” and “demonstrate” denote higher degrees of
certainty and these are usually reserved for strong scientific conclusions (i.e.,
less uncertainty, or less likelihood of an absence of an association). All four
levels could potentially be quantified with measures of “statistical significance,”
but the committee does not assign such measures in most instances because the
data at hand are generally not appropriate for such precise quantification of the
degree of uncertainty.
In summary, the reader is cautioned to consider carefully the terminology
in this report in light of the fact that the qualifiers of certainty used here are
those used most commonly in the scientific literature and that these words can
carry different weight in common language and in the courtroom.
3.3 THE FEDERAL COORDINATED RESPONSE AND
ASSIGNMENT OF LABORATORY WORK
Oversight and coordination of a complex scientific study are critical. Large
teams of scientists have been successful at complex studies (e.g., in particle
physics and genomic research) because they have a clear leadership structure for
the coordination and planning of their efforts (International Human Genome
Sequencing Consortium, 2001; Venter et al. 2001; NRC, 2003). With pressures
of time and expense in any study, someone or some group in the research team
must make decisions about which avenues to pursue and which to abandon.
In 2001, the FBI had a science laboratory at Quantico, the Hazardous
Materials Response Unit, and another team of weapons of mass destruction
(WMD) experts, but it did not have the capabilities to handle all of the types
of scientific experiments that would be required to examine the evidence in the
investigation of the B. anthracis mailings. FBI investigators quickly realized the
need to turn to outside laboratories and experts for help. The Bureau imme -
diately formed an internal group that had members from the scientific team,
investigative team, and terrorism team. Recognizing the importance of parallel
criminal and scientific investigations, the FBI embedded high-level DOJ staff
from the internal team, who then advised them throughout the investigation.
At that time, the FBI did not have the organizational structure needed to
oversee such a complex, multifaceted, and involved scientific investigation.
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56 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
In 2003, the agency remedied this organizational limitation by forming a new
unit focused on investigations involving chemical, biological, radiological, and
nuclear sciences, called the Chemical, Biological, Radiological, and Nuclear
(CBRN) Sciences Unit, or the Chemical Biological Science Unit (CBSU).
The committee was told that the team working on the scientific investi -
gation met weekly with the law enforcement team for information sharing,
strategy, and coordination. Reports and notes from some of these meetings
were shared with the committee late in the process of finalizing this report. The
reports indicate the complexity of the parallel tracks of the investigation and
document progress of each aspect of the scientific efforts and the decisions to
proceed with or abandon particular lines of the investigation.
In the early stages of the investigation, the FBI sought the advice of outside
experts to assist in characterizing the properties of the B. anthracis evidence.
The engagement of these experts was aided by the creation of an advisory group
led by the director of the National Science Foundation, the director of the
National Institute of Allergy and Infectious Diseases of the National Institutes
of Health, and federal officials from numerous other science agencies. This
group met regularly in classified sessions with FBI leaders to hear about the
investigation and to provide advice and the names of potential subject matter
experts the FBI could engage for assistance.
In addition, several Technical Review Panels were formed consisting of
scientists from Department of Energy National Laboratories, academic laborato-
ries, and members of the National Academy of Sciences. Panels were constituted
to review the analytical plan (that is, what tests should be done and by whom),
the progress of the investigation, and the chemistry and biology techniques used.
We reviewed several reports from meetings held in late 2001 and thereafter (FBI
Documents, B1M1, B3D1-7). According to these materials and the DOJ report,
“At the outset of the investigation, three panels comprised of 33 of the nation’s
leading authorities in bioweapons development from the former offensive bio -
weapons program, microbiology, chemistry, and microscopy were convened to
assist the FBI in developing a comprehensive analytical framework to evaluate
the anthrax powders recovered from the envelopes and the contamination found
in the AMI Building” (USDOJ, 2010, p. 13).
The FBI benefitted from these early informal and regular meetings of
senior leadership from other science agencies (FBI, 2009; Colwell, 2009). The
FBI also received input from the Department of Defense (DOD), the Intel -
ligence Community, DOJ, CDC, and Armed Forces Institute of Pathology
(AFIP) regarding the scientific investigation (FBI/USDOJ, 2011).
As is shown in Table 3-1, the U.S. Army Medical Research Institute of
Infectious Diseases (USAMRIID) played a central role in the scientific inves -
tigation. The facility had provided analytical services to the FBI Laboratory
since 1998. During October and November 2001, scientists at USAMRIID
were included in the team performing on-site testing at the American Media,
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57
SCIENTIFIC INVESTIGATION IN A LAW ENFORCEMENT CASE
Inc. (AMI) building in Florida, and they conducted the initial examinations of
the letter spore preparations for physical characteristics (using microscopy and
electron microscopy) and spore viability (see Chapters 4 and 5). USAMRIID
scientists also conducted microbiological analyses and identified the dominant
and variant morphological colony types that appeared in the evidentiary mate -
rial (see Chapter 5).
The FBI also sought the help of dozens of outside laboratories. In total,
thousands of samples were processed and analyzed by 29 academic, govern -
ment, and private BSL-3 laboratories across the country (Piggee, 2008; USDOJ,
2010). The work commissioned by the FBI was highly compartmentalized.
Most of the laboratories conducting analyses were not aware of other analyses
under way (Keim, 2009; Michael, 2009; Weber, 2009). Scientists at these labo -
ratories responded rapidly and provided the bulk of the scientific studies on
which the FBI relied in its investigation. The committee read reports prepared
by outside scientists responding to specific requests from the FBI and we
received reports of periodic reviews of contracted work by panels of experts
(B3D1-7). The committee also reviewed reports of work carried out in parallel
at the AFIP although it is not clear how closely AFIP and the FBI investigative
and scientific teams worked together or coordinated their efforts.
As the scientific investigation proceeded, several laboratories conducted
sequential and parallel scientific analyses on the evidentiary material gathered
from the letters, environmental samples, and clinical samples (see Table 3-2).
These analyses first focused on identifying the nature of the letter and environ -
mental materials, their similarities and differences, their biological, chemical,
and physical properties, and, eventually, their similarity to other samples of
Ames strain B. anthracis in laboratories around the world. According to the
affidavit in support of a search warrant submitted by Postal Inspector Thomas
F. Dellafera (Case number O7-524-M-01, October 31, 2007), 16 domestic
laboratories and three foreign laboratories (in Canada, Sweden, and the United
Kingdom) were identified as having the Ames strain in their inventories prior
to the attacks; two additional domestic laboratories were subjected to consent
searches, and one domestic laboratory was subjected to a search warrant. A
subpoena prepared by the FBI in early 2002 for sample submission specified
that only Ames strain samples be submitted; the subpoena protocol for these
submissions is described in Chapter 6. In the end, the FBI assembled a reposi-
tory of over 1,070 Ames strain samples from 20 laboratories, of which 1,059
were viable. Attributes of the samples in the repository were compared against
the characteristics of the evidentiary samples (as discussed in Chapters 5 and 6).
According to FBI officials, the focus of these analyses was to provide FBI
investigators with scientific leads that could be used to assist in its criminal
investigation (FBI, 2009). Chapters 4 through 6 provide in-depth descriptions
of the analyses conducted, conclusions reached, and this committee’s findings
and lessons learned for the future.
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64 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
by the FBI (see Figures 3-2 and 3-3). In brief, the powders from the New York
Post and Brokaw letters were multicolored and granular in consistency. The
envelopes in the second wave of mailings contained a powder of uniform color
and smaller particle size, which would facilitate more efficient airborne transmis-
sion of the B. anthracis spores and could account for the prevalence of inhalation
cases in the second cluster (Hassell, 2009). In addition, despite the fact that the
envelopes in the October mailings were unopened during their passage through
the postal centers, the use of high-speed processing and sorting machines may
have contributed to dispersal of the spores and exposure of postal workers.
Greene and colleagues (2002) reported that, although the envelopes containing
the B. anthracis were handled in only a small area of the Trenton facility, envi-
ronmental sampling found evidence of spores throughout the facility.
The B. anthracis isolates cultivated from the clinical specimens of patients,
the four recovered powder-containing envelopes, and over 100 environmental
samples collected along the suspected path traveled by the contaminated mail
were subtyped by the CDC using multiple-locus variable-number tandem repeat
analysis and sequencing of the protective antigen gene (pagA). In addition, pagA
was amplified and sequenced directly from some clinical specimens. All of the
results indicated the presence of the B. anthracis Ames strain (Hoffmaster et al.,
2002).
Jernigan and colleagues (2002) noted that their epidemiological investiga -
tion had several limitations. Because identification of case patients involved
numerous local, state, and federal officials, data collection methods were not
uniform. The widespread use of postexposure prophylaxis and the difficulty of
obtaining information about potentially exposed persons prevented accurate
estimates of anthrax exposure rates. Some cases may have been overlooked
because patients might have been administered antimicrobials after being mis -
takenly diagnosed with other types of infectious diseases, without physicians
recognizing the disease to be anthrax. The lack of prior experience with bio-
terrorism also forced the investigators to refine methods and redefine interven -
tions on a continuing basis.
3.4.2 Crime Scene Environmental Samples
The FBI hazardous materials (HAZMAT) team, with the assistance of
scientists from USAMRIID, CDC, USPIS, Environmental Protection Agency
(EPA), and contractors, performed environmental investigations to assess the
presence and extent of B. anthracis contamination and to guide decontamina-
tion and environmental remediation (Jernigan et al., 2002). Environmental
samples were collected at contaminated worksites and mailboxes by public
health, law enforcement, and other government and contract staff (CDC,
2001a; Sanderson, 2001, 2002). At the time, the FBI did not have biosafety
level 3 (BSL-3) laboratory facilities capable of handling B. anthracis, so samples
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SCIENTIFIC INVESTIGATION IN A LAW ENFORCEMENT CASE
were tested at laboratories participating in the local, state, and federal investiga-
tion efforts as described in Chapters 4 through 6.
Crime scene environmental samples were collected by surface sampling,
mostly with RODAC (replicate organism detection and counting) contact
plates. Swabs, wipes, high-efficiency particulate air (HEPA) vacuum filtration,
and air sampling also were used (Dull et al., 2002; Jernigan et al., 2002; Teshale
et al., 2002; Beecher, 2006). Based on the documents provided to the com -
mittee, it appears that the FBI and CDC relied on culture-based techniques
to detect B. anthracis in the environmental samples (Jernigan et al., 2002;
Hoffmaster et al., 2002). In much of the work on samples collected from the
environment, colonies propagated on agar plates were presumptively identi -
fied as B. anthracis based on morphology (Beecher, 2006). Selected colonies
were definitively identified as B. anthracis using standard confirmatory tests. At
least one environmental swab sample from AMI was sent to Patricia Worsham
at USAMRIID (in June 2005) for detection and identification of B. anthracis
variant colony morphotypes. Material from this swab was used to inoculate
sheep blood agar. The report of this work by Worsham (2009; FBI Docu-
ments, B1M2D14) states that B. anthracis variant morphotypes A, B, and C/D
were found, but not morphotype E, in addition to the wild-type colony mor-
photype. Furthermore, Worsham states that a Bacillus strain was recovered
that resembled the B. subtilis found in the New York Post letter. The report
states that in October 2006, cell suspensions from 34 colonies that exhibited
a variant morphotype, as well as from the B. subtilis-like isolate, were sent to
the National Bioforensic Analysis Center (NBFAC) for DNA extraction, and
that the DNA from the variant colony morphotypes were to be sent to the
Institute for Genomic Research (TIGR) for sequencing of the morphotype A,
B, C/D, and E genomic regions. However, according to statements by the FBI
to this committee (FBI/USDOJ, 2011), the U.S. Attorney’s Office advised that
this sequencing and further characterization of these colony morphotypes from
AMI would not be undertaken.
The FBI did not use molecular assays for detecting B. anthracis DNA directly
in environmental samples (in the absence of a cultivated isolate). Molecular assays
targeting B. anthracis-specific genetic markers had been developed prior to 2001
for detecting this organism in food (Yamada et al., 1999) and in environmental
samples (Beyer et al., 1995). An on-site polymerase chain reaction (PCR)-based
device was used by the CDC at the Brentwood postal facility in 2001 for pre-
liminary assessment or adjunct analysis of B. anthracis DNA in environmental
samples, but this approach had not been validated by them at that time for these
types of samples. However, the Biological Aerosol Sentry and Information System
was deployed in 2001 for environmental monitoring and incorporated PCR-based
detection methods for a variety of biological agents (Fitch et al., 2003).
The committee recognizes that the FBI may have hesitated to apply a newly
emerging method to the assessment of forensic evidence before it had been widely
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66 SCIENTIFIC APPROACHES USED TO INVESTIGATE THE ANTHRAX LETTERS
adopted or validated for this purpose; however, under exigent circumstances, it
can be appropriate to proceed without completing full validation. The FBI stated
to the committee (FBI/USDOJ, 2011) that laboratories in the CDC Laboratory
Response Network that were equipped for PCR analysis were overwhelmed with
samples early in the investigation, and that the FBI obtained more timely results
on the presence of B. anthracis in environmental samples by relying instead on
RODAC plate isolation techniques. In any case, by 2004, PCR had been validated
for various bacterial agents in environmental samples (Malorny et al., 2004) and
could have been performed on the environmental samples from 2001.
In addition to the environmental sampling, nasal swab specimens were col-
lected from potentially exposed individuals to help delineate the area of expo -
sure to aerosolized spores and to determine where persons with inhalational
anthrax might have been exposed based on where they worked. According to
Jernigan and colleagues (2002), because the sensitivity of nasal swab cultures
diminishes with time following human exposure, attempts were made to obtain
cultures within seven days of exposure. The presence of B. anthracis from nasal
swab cultures was determined by Gram stain and colony characteristics as well
as through confirmatory testing by laboratories participating with the local,
state, and federal efforts (Jernigan et al., 2002).
Finally, in the new materials provided to the committee it is noted that PCR
analysis was performed on human remains from United flight 93 on 9/11/2001
that were identified as those of the hijackers (B3D1). Analysis was performed
at USAMRIID and at AFIP for sequences diagnostic of B. anthracis. One assay
at USAMRIID gave positive results, but these results were believed by the FBI
to be due to laboratory contamination. All other results were negative. As the
committee learned at the January 2011 meeting, there were no tests done on
remains from any of the other September 11, 2001 hijackers.
3.4.3 Samples from an Overseas Site Identified by Intelligence
In December 2010-January 2011, the FBI first made available to the Com-
mittee “AMX Weekly Science Updates” and a newly de-classified document
that described the collection and analysis of environmental samples from an
undisclosed site outside the continental United States (OCONUS) for the
presence of B. anthracis Ames (FBI/USDOJ, 2011, FBI Documents, WFO
Report). This work was performed as part of the anthrax letters investigation.
Few details were made available to the committee.
At least three sample collection missions were conducted by the FBI and/or
partners from the intelligence community at an overseas site because of informa-
tion about efforts by Al Qaeda to develop an “anthrax program” (FBI/USDOJ,
2011). In May 2004, the FBI and partners from the intelligence community
visited an overseas location at which they had been told an anthrax program
had been operating, and brought back swab and swipe samples to the United
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States. None of the samples grew B. anthracis after incubation in culture media.
However, three swab samples were reported as positive for B. anthracis and for
B. anthracis Ames-specific sequences by PCR, including swabbings from the
outside of an unopened medicine dropper package, a sink, and a sink drain hose.
Repeat testing of these three positive samples as part of a group of 15 blinded
samples, including soil samples, water blanks and non-Ames Bacillus species,
again yielded positive results for two of the three same samples (and for none
of the other samples). However, not all replicates of the DNA extracts from the
positive samples gave positive results. Apparently, an earlier collection mission
to this site, prior to May 2004, by others in the intelligence community had also
yielded samples with positive PCR results for B. anthracis DNA and negative
culture results. As a result of these findings, a third collection mission was con-
ducted in November 2004 and this time large portions of the site were returned
intact to the United States, including the entire sink, drain, and associated
plumbing that had been the source of the positive March 2004 samples. These
items were extensively sampled, and again tested for both viable B. anthracis and
for B. anthracis DNA. This time, according to the June 2008 declassified docu-
ment, all the tests were negative (FBI Documents, WFO Report).
The committee was provided only fragmentary information about and
limited primary data from this work and received them very late in our study.
We consider these data to be inconclusive regarding the possible presence of B.
anthracis Ames at this undisclosed overseas site. Several scientific and technical
issues should be explored in more detail, such as the performance character-
istics of the assays, whether or not the assays were validated for use with these
sample types, the degree to which samples or sample locations gave repeatedly
positive results, interpretation of inconsistent positive results, whether or not
the Ames genetic mutations in the anthrax letters were detected in any of these
overseas samples, and the natural distribution of B. anthracis strain types in this
overseas geographic region.
3.4.4 Letter Material and Cross Contamination
Material was collected directly from the Daschle, Leahy, New York Post,
and Brokaw letters, but in varying quantities. As indicated above, no letter was
found at the AMI building in Florida, thus only environmental and clinical
samples were available for analysis. The decision by the U.S. Attorney’s Office
not to pursue molecular analysis on the AMI crime scene samples (FBI, 2011)
limits the ability to definitively connect this attack to the material in the recov -
ered letters from New York and Washington, D.C.
The letter addressed to Tom Brokaw at NBC was found after Erin
O’Connor, an assistant to Brokaw, developed cutaneous anthrax after opening
a letter containing a white powder (Cole, 2009). As mentioned before, the FBI
laboratory was not equipped to handle B. anthracis, so all of the New York
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samples were sent to the New York Department of Health in Albany or else -
where. The New York City Health Department officials who tested the Brokaw
letter accidentally lost most of the sample and contaminated the laboratory,
rendering the space unusable for a critical period of time after the B. anthracis
was discovered and before the full scope of the problem was known. As a result,
little material from the Brokaw letter remained available for further analyses. A
biopsy obtained from a black eschar lesion that formed on O’Connor and was
sent to CDC tested positive by immunohistochemical staining for the cell wall
antigen of B. anthracis.
The New York Post letter had been thrown into a bin for hate mail and was
found after employee Johanna Huden became ill with cutaneous anthrax. The
letter had been unopened and the enclosed material was available for future
analyses.
On October 15, when staff in Senator Daschle’s office opened an envelope
containing a white powder, police quarantined the office and surrounding
rooms, shut down the Capitol’s mail system, and suspended public tours. Law
enforcement officials in protective biohazard suits took over Senator Daschle’s
office. Unlike previous samples, which were sent to the CDC for analysis, the
FBI sent the Daschle letter to USAMRIID because of its biocontainment facili -
ties (FBI, 2009). Since much of the material was dispersed when the letter was
opened, limited material was available for future analyses.
On November 16, 2001, FBI and EPA HAZMAT personnel found the
letter addressed to Senator Leahy in one of 280 barrels of unopened mail col -
lected from Capitol Hill after the discovery of the anthrax-contaminated letter
sent to Senator Daschle (FBI, 2008a). The search that resulted in retrieval of the
Leahy letter was conducted by teams of HAZMAT workers from the FBI and
EPA Criminal Investigative Division (FBI, 2008a). They developed a sampling
protocol intended to eliminate the need for HAZMAT teams to sift through
each piece of mail in 642 trash bags to find contaminated mail (FBI, 2008a).
The mail was sampled and sorted in a containment facility constructed in
a large warehouse and maintained with negative air pressure. The intake and
exhaust air was passed through HEPA filters, which trap essentially all particles
the size of anthrax spores. Air samplers were used to monitor the air inside
and outside the containment area for the presence and quantity of airborne
spores. Investigative workers were monitored for exposure by sampling their
clothing. In general, the only investigators who were contaminated were those
who handled a “hot” bag containing the letter addressed to Leahy laden with
spores (Beecher, 2006).
Each bag was shaken in an attempt to distribute any spores that could
be present. A swab was then inserted into a small hole in each bag and wiped
around the inside. After the swab was withdrawn, the hole was sealed with duct
tape and the swab was used to inoculate a Petri dish, which was sent to the
Naval Medical Research Center (NMRC) for analysis (FBI, 2010b).
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Trace contamination was detected in 62 bags, likely due to a high level of
shedding from cross-contaminated mail (Beecher, 2006). Five bags produced
more than 100 bacterial colonies from a swab and were considered “hot.”
Innovative air sampling was used to maximize the recovery of the target organ -
ism (Beecher, 2010). One bag, the one containing the Leahy letter, was orders
of magnitude more contaminated than the others. This bag produced between
19,000 and 23,000 spores (or 760 to 920 colony-forming units per liter of air
sampled) (Beecher, 2006).
Beecher (2006) examined some of the issues related to cross contamina-
tion of mail, mail bags, and the local environment by the Leahy letter. He
documented extensive contamination of personnel especially during physical
handling of the spore-laden letter. In the conduct of this work, the investiga -
tors followed thoughtful and appropriate practices and procedures for the
purpose of minimizing artifactual cross contamination. Of note, they identi -
fied a correlation between the degree of letter contamination and the previous
handling of the letter by sorting machines in the Trenton and Washington,
D.C., postal processing and distribution centers. The insights from this work
proved useful in developing a more detailed understanding of the route taken
by the anthrax letters from the point of deposit through the U.S. Postal System
to the site of delivery. Although suggestive of a mechanism and scenario by
which Ottilie Lundgren, a resident of Oxford, Connecticut, might have devel -
oped inhalational anthrax (cross contamination of mail en route to her home),
the committee lacked sufficient information (e.g., other possible exposures,
unusual susceptibility to low numbers of anthrax spores) with which to assess
the plausibility and likelihood of this mechanism and this particular scenario.
On September 1, 2001, Defence Research Establishment Suffield (DRES)
in Canada released the results of a study (FBI Documents, B2M11D1) that had
been designed to measure and better understand the dispersion of spores that
might occur after the opening of an envelope containing B. anthracis spores.
This study involved a series of experiments in which envelopes containing
either 0.1 or 1.0 gram of B. globigii spores at a concentration of ~1 × 1011 cfu/g
(as a surrogate for B. anthracis spores) were opened in a DRES aerosol test
chamber that was configured to represent a mail room or office. The chamber
measured 18 × 10 × 10 ft (i.e., with a volume of 1,800 cu ft) and had a recircu -
lating air handling system operating at 1,050 cu ft/min. The presence of spores
at various sites in the chamber was assessed using culture-based approaches,
not molecular detection methods. The results showed that the act of handling
or opening these envelopes was “far more effective than initially suspected” in
causing dispersion of spores in the chamber. Particles of respirable size were
released quickly and spread throughout the chamber, such that after the open -
ing of a 0.1 g spore envelope, 10 minutes of exposure to the air in the chamber
would have provided a dose 480-fold greater than the amount needed to kill a
human with 50 percent probability. The investigators noted that envelopes were
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more likely to cause cross contamination of the local environment, including
the envelope handler, if the open corners of the envelope were not deliberately
sealed by the preparer. The investigators at DRES did not seal the corners of
the envelopes they used in these experiments. However, the corners of the
envelopes mailed through the U.S. Postal System with B. anthracis spores in
September and October 2001 apparently were sealed. This study was valuable
in revealing the potential speed, magnitude, and spatial distribution of envi -
ronmental contamination by spores subsequent to the handling or opening of
a spore-laden envelope.
3.5 COMMITTEE FINDINGS AND RECOMMENDATIONS
The events of autumn 2001 unfolded rapidly, with CDC’s initial public
health response quickly extended to a major criminal investigation under the
control of the FBI. A multipronged investigative strategy emerged for the sci -
entific investigation, with one set of activities focused on understanding the
characteristics of the material in the letters and another on developing and
conducting a comparative analysis of these evidentiary materials against samples
collected from the scientific community. The science and technology that formed
the basis for these analyses evolved rapidly and had a major impact on the field
of microbial forensics. Although the public health crisis largely subsided after
the last victim died in November 2001, the scientific investigation continued
until 2008 and the criminal investigation continued until the case was closed in
February 2010 (see Table 3-1).
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 regu -
larly 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.
In a complex investigation during a period of extreme national urgency
such as this one, it is imperative to recruit and make wise use of the best and
most relevant expertise. The Bureau regularly briefed its high-level advisory
group in a secure setting where classified material was reviewed. They also
relied on subject matter experts and expertise from other government agen-
cies. The committee recognizes, in agreement with the FBI (FBI/USDOJ, Jan
2011) that the unique skills and expertise needed to conduct microbial forensic
examinations might, in some cases such as this one, require reliance on the same
set of scientific personnel, who also might be considered potential suspects.
Thus it is important to have in place an external oversight structure with the
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capacity to recognize where relevant expertise and conflicts might reside and
to provide guidance and coordination for the overall scientific investigation. In
addition, the small number of laboratories with expertise in a given pathogen or
technique requires that a certain level of independent oversight be maintained.
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.
Weekly meetings occurred between the science team and those leading the
criminal investigation. The results of these meetings and the way the scientific,
criminal and legal aspects of the investigation interplayed will be beneficial in
preparing for future attacks (B3D1). In addition, the development of a new
U.S. Government microbial forensics infrastructure and research strategy will
be important steps toward enhancing future capabilities for attribution in the
event of a biological attack (Pesenti, 2010).
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.
The committee recognizes that the circumstances of 2001 created an
abundance of samples and associated work for the LRN (laboratory response
network) resulting in a decision to use RODAC plates for analysis of the envi -
ronmental samples. Thus, the FBI did not confirm the presence of the letters-
associated genetic mutations in the environmental samples. In the nine years
since that time, dramatic advances in high throughput sequencing technology
have greatly improved the ability to detect and characterize rare strains and
species in complex environmental samples2 (for further, related discussion,
2 Multiple studies in each of a number of diverse environmental settings have demonstrated the
feasibility and reliability of resolving strain-specific fine genomic structure (at the level of SNPs
and local structural rearrangements) in highly complex biological samples using next-generation
sequencing technology and metagenomic approaches. Examples include strain resolution and gene
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see Finding 6.8 in Chapter 6). In thinking of future incidents, it is imperative,
based on lessons learned from this investigation, to anticipate the types of situ -
ations and circumstances that might affect evidence collection, preservation,
and documentation, and to employ protocols and procedures that ensure the
best possible outcomes.
Finding 3.4: There was inconsistent evidence of B. anthracis Ames DNA in
environmental samples that were collected from an overseas site.
At the end of this study, the NRC 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 investigation. The
information indicates that there was inconsistent evidence of Ames strain
DNA in some of these samples, but no culturable B. anthracis. The commit-
tee believes that the complete set of data and conclusions concerning these
samples, including all relevant classified documents, deserves a more thorough
scientific review.
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).
The scientific investigation of any future biological attack would greatly
benefit fromrobust independent oversight and ongoing review. To accomplish
this, the government should maintain a standing body of scientific experts with
proper security clearances who are fully briefed on matters of importance for
preparedness and response to a biological attack. When an investigation is
launched, members of this group could help guide the scientific investigation.
In preparing for future investigations, all relevant U.S. government agen -
cies and departments will need to work together to ensure that independent,
family structure in ocean surface water (see multiple publications from the Sorcerer II Global Ocean
Sampling Expedition), evidence for rare strain ecotypes in acid mine drainage (see multiple publi -
cations from the Banfield group at UC Berkeley), and gene family sequence microheterogeneity in
prophage genomes that have been reconstructed from shotgun sequencing of virus-like particles
in human feces (e.g., see supplemental data from Nature 466:334, 2010). This large and growing
body of work strongly suggests that the application of these same techniques to some of the envi -
ronmental samples from the anthrax letters case might provide additional clarity about B. anthracis
genome sequence variants and the relationships among the strains in the samples and the letters.
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high-quality, external science advice is available from individuals with expertise
in critical scientific areas likely to be relevant given anticipated scenarios for
scientific investigations. By identifying a core set of external experts and con -
vening this group ahead of time, productive working relationships could be
established between this group and members of the government’s bioforensics
community. When a new investigation is launched, this core set of external
experts could assist in recruiting others with more specific expertise relevant
to the investigation under way. For example, the FBI included some expert
scientists early in the anthrax letters investigation, but it does not seem to have
sought formal expertise in statistics until the investigation was nearly com -
pleted. Because many inferences depend on the design and analysis of datasets
that may be complex, as in this case, for any similar investigation in the future,
it will be important that the FBI consult with expert statisticians throughout
the processes of experimental design and planning, sample collection, sample
analysis, and data interpretation. The committee recognizes that much has been
done by the government over the past several years to build and enhance this
important infrastructure and set of resources, although the scope of this study
did not include assessment of the current infrastructure.
When an investigation is launched, the panel of external experts could
recommend and review strategies, protocols, and procedures; help with the
development of new methods and scientific approaches; provide advice on
the selection of contract scientists and additional outside experts; assist in data
interpretation; and help generate alternative hypotheses. Members of the panel
should not be directly involved in conducting the scientific investigation itself.
It will also be important that the panel have an ongoing role throughout the
course of the investigation and be briefed on all of the science that is contem -
plated and pursued. Consistent documentation of recommendations and input,
and the subsequent responses, will be of great value.
In the future relevant agencies should review and periodically update
appropriate protocols and experimental designs to use best strategies for pre -
serving evidence, exploiting samples for scientific information, and meeting
subsequent legal challenges. Protocols should ensure that clinical and envi -
ronmental evidence is properly collected, preserved, documented, and ana -
lyzed to maximize the utility of the samples collected. Furthermore, state-of-
the-art molecular methods, such as next-generation nucleic acid sequencing
techniques, offer great potential for characterizing clinical and environmental
samples (see Finding 6.8 in Chapter 6).
Recommendation 3.1: A review should be conducted of the classified materi -
als 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 collections.
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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 and the importance we placed
on issuing a timely report, and the agreement between the NRC and the FBI
that all material we considered be publicly available, the committee did not
undertake this additional review of classified material.
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.
Communicating with the public and policymakers is extremely impor-
tant in order to ensure that accurate information is available and to minimize
unrealistic expectations. Special attention will need to be paid to communicat -
ing scientific information to these groups in an accurate and credible manner,
especially if the information will play a critical role in the investigation.
When presenting to the public the findings of an investigation that involve
scientific evidence, especially one as important as the anthrax letters investiga -
tion, officials will need to make every effort to have scientists verify the accuracy
of the scientific information they report. The inaccurate reporting of facts or
the overstatement of scientific evidence is a disservice to the public. In the
anthrax letters investigation, there were repeated claims that all of the attack
letters contained all of the genotypic variants (see Chapter 6 and Finding 6.7)
that implicated flask RMR-1029 as the source of the anthrax spores, when in
fact not all of the letters were checked for these variants. Of even greater con -
cern, because it suggested possible deception by the suspect, the strength of
the evidence was overstated that a disputed sample submitted by the suspect
had not come from the proper source (see Chapter 6 and Finding 6.4). Similar
mistakes can be avoided in the future by involving the relevant scientists in fact
checking of the reports before they are released.
