REVIEW OF THE
SCIENTIFIC APPROACHES USED
DURING THE FBI’S INVESTIGATION OF THE
2001 ANTHRAX LETTERS
Committee on Review of the Scientific Approaches Used
During the FBI’s Investigation of the 2001 Bacillus anthracis Mailings
Board on Life Sciences
Division on Earth and Life Studies
Committee on Science, Technology, and Law
Policy and Global Affairs Division
NATIONAL RESEARCH COUNCIL
OF THE NATIONAL ACADEMIES
THE NATIONAL ACADEMIES PRESS
Washington, D.C.
THE NATIONAL ACADEMIES PRESS 500 Fifth Street, N.W. Washington, DC 20001
NOTICE: The project that is the subject of this report was approved by the Governing Board of the National Research Council, whose members are drawn from the councils of the National Academy of Sciences, the National Academy of Engineering, and the Institute of Medicine. The members of the committee responsible for the report were chosen for their special competences and with regard for appropriate balance.
This study was supported by Contract No. A9N0902700 between the National Academy of Sciences and the U.S. Federal Bureau of Investigation. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the organizations or agencies that provided support for the project.
International Standard Book Number-13: 978-0-309-18719-0
International Standard Book Number-10: 0-309-18719-2
Library of Congress Catalog Card Number 2011927648
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THE NATIONAL ACADEMIES
Advisers to the Nation on Science, Engineering, and Medicine
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COMMITTEE ON REVIEW OF THE SCIENTIFIC APPROACHES USED DURING THE FBI’S INVESTIGATION OF THE 2001 BACILLUS ANTHRACIS MAILINGS
ALICE P. GAST (Chair), President, Lehigh University
DAVID A. RELMAN (Vice Chair), Thomas C. and Joan M. Merigan Professor, Stanford University School of Medicine, and Chief, Infectious Disease Section, Veterans Affairs Palo Alto Health Care System, CA
ARTURO CASADEVALL, Leo and Julia Forchheimer Professor of Microbiology and Immunology and Chair, Department of Microbiology and Immunology, Albert Einstein College of Medicine
NANCY D. CONNELL, Professor of Medicine, University of Medicine and Dentistry of New Jersey (UMDNJ)-New Jersey Medical School and Director, UMDNJ Center for BioDefense
THOMAS V. INGLESBY, Chief Executive Officer and Deputy Director of the Center for Biosecurity of University of Pittsburgh Medical Center, Associate Professor of Medicine and Public Health, University of Pittsburgh Schools of Medicine and Public Health
MURRAY V. JOHNSTON, Professor of Chemistry and Biochemistry, University of Delaware
KAREN KAFADAR, James H. Rudy Professor of Statistics and Physics, Indiana University
RICHARD E. LENSKI, John A. Hannah Distinguished Professor of Microbial Ecology, Michigan State University
RICHARD M. LOSICK, Maria Moors Cabot Professor of Biology, Harvard College Professor and Howard Hughes Medical Institute Professor
ALICE C. MIGNEREY, Professor of Chemistry and Biochemistry, University of Maryland, College Park
DAVID L. POPHAM, Professor of Microbiology, Virginia Polytechnic Institute and State University
JED S. RAKOFF, United States District Judge, Southern District of New York
ROBERT C. SHALER, Director, Forensic Science Program, Professor of Biochemistry and Molecular Biology, Pennsylvania State University
ELIZABETH A. THOMPSON, Professor of Statistics, University of Washington
KASTHURI VENKATESWARAN, Senior Research Scientist, California Institute of Technology Jet Propulsion Laboratory
DAVID R. WALT, Robinson Professor of Chemistry and Professor of Biomedical Engineering, Tufts University and Howard Hughes Medical Institute Professor
Staff
ANNE-MARIE MAZZA, Study Director
FRANCES E. SHARPLES, Study Co-Director (until 1/11)
ERICKA MCGOWAN, Program Officer, Board on Chemical Sciences and Technology (until 4/10)
STEVEN KENDALL, Associate Program Officer, Committee on Science, Technology, and Law
AMANDA P. CLINE, Senior Program Assistant, Board on Life Sciences (until 5/10)
KATHI E. HANNA, Consultant Writer
CAMERON H. FLETCHER, Editor
BOARD ON LIFE SCIENCES
KEITH R. YAMAMOTO (Chair), Executive Vice Dean, School of Medicine, and Professor, Department of Cellular and Molecular Pharmacology, University of California, San Francisco
BONNIE L. BASSLER, Investigator, Howard Hughes Medical Institute, and Professor of Molecular Biology, Department of Molecular Biology, Princeton University
VICKI L. CHANDLER, Chief Program Officer, Science, Gordon and Betty Moore Foundation
SEAN EDDY, Group Leader, Howard Hughes Medical Institute Janelia Farm Research Campus
MARK D. FITZSIMMONS, Associate Director, MacArthur Fellows Program, John D. and Catherine T. MacArthur Foundation
DAVID R. FRANZ, Vice President and Chief Biological Scientist, Midwest Research Institute
DONALD E. GANEM, Director, Global Infectious Disease Research, Novartis Institute of Biomedical Research
LOUIS J. GROSS, Professor of Ecology and Evolutionary Biology and Mathematics and Director, Institute for Environmental Modeling, Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville
JO HANDELSMAN, Howard Hughes Medical Institute Professor, Yale University
CATO T. LAURENCIN, Vice President for Health Affairs and Dean, University of Connecticut Health Center School of Medicine
BERNARD LO, Professor of Medicine and Director of the Program in Medical Ethics, University of California, San Francisco
ROBERT M. NEREM, Institute Professor and Parker H. Petit Professor Emeritus, Institute for Bioengineering and Bioscience, Georgia Institute of Technology
CAMILLE PARMESAN, Associate Professor of Integrative Biology, Section of Integrative Biology, University of Texas
MURIEL E. POSTON, Dean of Faculty, Skidmore College
ALISON G. POWER, Professor of Ecology and Evolutionary Biology and Dean, The Graduate School, Cornell University
BRUCE W. STILLMAN, President, Cold Spring Harbor Laboratory
CYNTHIA WOLBERGER, Professor, Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine
MARY WOOLLEY, President and CEO, Research!America
Staff
FRANCES E. SHARPLES, Director
JO L. HUSBANDS, Scholar/Senior Project Director
JAY B. LABOV, Senior Scientist/Program Director for Biology Education
KATHERINE W. BOWMAN, Senior Program Officer
MARILEE K. SHELTON-DAVENPORT, Senior Program Officer
INDIA HOOK-BARNARD, Program Officer
ANNA FARRAR, Financial Associate
CARL-GUSTAV ANDERSON, Program Associate
AMANDA MAZZAWI, Senior Program Assistant
AYESHA AHMED, Program Assistant
COMMITTEE ON SCIENCE, TECHNOLOGY, AND LAW
DAVID KORN (Co-Chair), Vice Provost for Research, Harvard University
RICHARD A. MESERVE (Co-Chair), President, Carnegie Institution for Science, and Senior Of Counsel, Covington & Burling LLP
FREDERICK R. ANDERSON, JR., Partner, McKenna, Long & Aldridge LLP
ARTHUR I. BIENENSTOCK, Special Assistant to the President for Federal Research Policy and Director, Wallenberg Research Link, Stanford University
BARBARA E. BIERER, Professor of Medicine, Harvard Medical School, and Senior Vice President, Research, Brigham and Women’s Hospital
ELIZABETH H. BLACKBURN, Morris Herzstein Professor of Biology and Physiology, University of California, San Francisco
JOHN BURRIS, President, Burroughs Wellcome Fund
ARTURO CASADEVALL, Leo and Julia Forchheimer Professor of Microbiology and Immunology; Chair, Department of Biology and Immunology; and Professor of Medicine, Albert Einstein College of Medicine
JOE S. CECIL, Project Director, Program on Scientific and Technical Evidence, Division of Research, Federal Judicial Center
ROCHELLE COOPER DREYFUSS, Pauline Newman Professor of Law and Director, Engelberg Center on Innovation Law and Policy, New York University School of Law
DREW ENDY, Assistant Professor, Bioengineering, Stanford University, and President, The BioBricks Foundation
PAUL G. FALKOWSKI, Board of Governors Professor in Geological and Marine Science, Department of Earth and Planetary Science, Rutgers, The State University of New Jersey
MARCUS FELDMAN, Burnet C. and Mildred Wohlford Professor of Biological Sciences, Stanford University
ALICE P. GAST, President, Lehigh University
JASON GRUMET, President, Bipartisan Policy Center
GARY W. HART, Wirth Chair in Environmental and Community Development Policy, University of Colorado, Denver
BENJAMIN W. HEINEMAN, JR., Senior Fellow, Harvard Law School and Harvard Kennedy School of Government
D. BROCK HORNBY, Judge, U.S. District Court, District of Maine
ALAN B. MORRISON, Lerner Family Associate Dean for Public Interest and Public Service, George Washington University Law School
PRABHU PINGALI, Deputy Director of Agricultural Development, Global Development Program, Bill and Melinda Gates Foundation
HARRIET RABB, Vice President and General Counsel, Rockefeller University
BARBARA JACOBS ROTHSTEIN, Director, The Federal Judicial Center
JONATHAN M. SAMET, Professor and Flora L. Thornton Chair, Department of Preventive Medicine, Keck School of Medicine and Director, Institute for Global Health, University of Southern California
DAVID S.TATEL, Judge, U.S. Court of Appeals for the District of Columbia Circuit
SOPHIE VANDEBROEK, Chief Technology Officer and President, Xerox Innovation Group, Xerox Corporation
Staff
ANNE-MARIE MAZZA, Director
STEVEN KENDALL, Associate Program Officer
Preface
In autumn 2001, the tragic deaths, illnesses, and environmental contamination caused by the mailing of Bacillus anthracis (B. anthracis) spores in letters sent through the U.S. postal system caused tremendous fear and disruption in a nation shaken by the events of September 11. Efforts led by the Federal Bureau of Investigation (FBI) to characterize the material contained in the letters and identify the individual or individuals responsible for the mailings would involve extensive scientific study spanning almost nine years.
It is not unusual to use science to identify and characterize evidence and to link it to a particular individual in a criminal investigation. Indeed, in the 2001 B. anthracis mailings investigation, physics, chemistry, and biology all played a role. In this case, the field of bacterial genomics was rapidly evolving throughout the investigation. Recognizing the challenges inherent in such a complex scientific investigation, in 2008 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 its investigation. In 2009, the committee, formed under the auspices of the National Academies’ Board on Life Sciences and Committee on Science, Technology, and Law, began this review.
As we undertook this review, the committee kept in mind the context of the time, immediately following September 11, 2001, when there were multiple high-profile FBI investigations under way. We recognized that the grave consequences of these events for public health and national security and the uncertainty about possible additional attacks necessarily influenced the initial design and execution of the FBI’s scientific investigation. Throughout its investigation, the FBI embraced and energetically pursued the use of new and emerging science through an unusual degree of involvement of outside scientists. In many ways, this case established and emphasized the potential importance of microbial forensics in the investigation of future acts of bioterrorism.
A scientific study is much more than a series of well-executed experiments. The planning and decision making used during a study are essential compo-
nents of the science and can determine its outcome. As we learned from our review of this case, it is especially important in emergency situations to have a clearly defined approach for undertaking a complex scientific investigation involving many experts and collaborators. In any such case, the goal is to integrate a broad range of experimental methods and exploratory work with a more thorough and deeper study of selected methods, along with clear guidance for the investigation. Investigators must maintain a healthy degree of skepticism and a willingness to challenge their own assumptions. Achieving these goals requires considerable thought and planning before a crisis occurs. It is also important to recognize that when science meets law enforcement there are several tensions that need to be balanced: openness and secrecy, collaboration and independence, and deliberateness and expediency.
We also learned from this investigation that there is an immediate and ongoing need from the outset of an investigation to obtain expert advice and have available a group of advisors who can provide conceptual insight and relevant expertise to scientific plans, approaches, and scenarios.
An unavoidable observation from the 2001 B. anthracis mailings is that the best subject matter experts in a given area also might be viewed as suspects. Working with potential suspects during a sensitive investigation is a challenge that the law enforcement community must continually address through its vetting processes.
Throughout our review, we focused on the scientific aspects of the investigation and did not evaluate non-science-based investigative material. We have evaluated the science to the best of our ability, given the materials made available to us. While there may be additional relevant material to which we were not provided access, we believe that our review of the available material has resulted in many useful findings and conclusions. Nonetheless, other aspects of, and documents from, the FBI investigation may deserve future study and review.
In following our charge, we evaluated the specific conclusions drawn by the FBI based on its scientific analyses. The FBI never provided the committee with those conclusions in written form, although FBI conclusions were offered in oral presentations to the committee. We repeatedly sought written statements of conclusions until the case was closed by the Department of Justice (DOJ) on February 19, 2010. In our report, we address the conclusions offered in verbal reports, as well as the main scientific conclusions as written in the DOJ Amerithrax Investigative Summary.1
In November 2010, after our final report had been submitted to the FBI for a security review, the FBI informed the National Academies that there were additional materials relevant to the committee’s work that had not previously
__________
1 United States Department of Justice. Amerithrax Investigative Summary. February 19, 2010. Available at: www.justice.gov/amerithrax/docs/amx-investigative-summary.pdf.
been shared with the committee. The Bureau offered to provide our committee with these materials and an additional briefing. After serious discussions with the National Academies’ leadership, we agreed to receive and review these materials and reconvene the committee for one final meeting in January 2011. The documents and briefing provided us with additional information and led to meaningful changes in this report regarding the organization of the scientific investigation, sample collection, and analytical tests undertaken by the FBI and its contracting laboratories. This information resulted in the addition of a new section in the report (3.4.3) and the addition of a new finding (3.4) and recommendation (3.1). A benefit of the extension of the project and the delay in issuance of the report is that important additional materials, now available to the public, provide more information about the scientific investigation.
LOOKING TO THE FUTURE
While much of our effort was focused on a review of the science performed in support of the investigation of the 2001 B. anthracis mailings, an equally important aim has been to help ensure that future scientific investigations of biological attacks are conducted in the most relevant, rigorous, 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 also may need to manage expectations among the general public, policymakers, and the scientific community about the conclusions that can realistically be expected from the use of microbial forensics.
We have been fortunate to work with extremely talented, intelligent, and dedicated individuals in the undertaking of this multifaceted study. Committee members evaluated large numbers of documents under constrained circumstances that required exceptional dedication and patience. They listened intently to speakers, asked probing and insightful questions, and vigorously discussed what was learned, what we could research, and how to word our findings. We are indebted to them for all the time and energy they gave to this effort. We are also most grateful to the staff—Amanda Cline, Cameron Fletcher, Steven Kendall, Ericka McGowan, Anne-Marie Mazza, and Fran Sharples—and to the consultant writer, Kathi Hanna.
Alice P. Gast and David A. Relman
Chair and Vice Chair
Acknowledgments
This report has been reviewed in draft form by individuals chosen for their diverse perspectives and technical expertise, in accordance with procedures approved by the National Research Council’s Report Review Committee. The purpose of this independent review is to provide candid and critical comments that will assist the institution in making its published report as sound as possible and to ensure that the report meets institutional standards for objectivity, evidence, and responsiveness to the study charge. The review comments and draft manuscript remain confidential to protect the integrity of the process. We thank the following individuals for their review of this report:
R. John Collier, Harvard Medical School
Rita R. Colwell, University of Maryland
M. Bonner Denton, University of Arizona
Ashlee M. Earl, Broad Institute of the Massachusetts Institute of Technology and Harvard University
Philip C. Hanna, University of Michigan Medical School
Stephen A. Johnston, Arizona State University
David H. Kaye, Arizona State University
Cato T. Laurencin, University of Connecticut Health Center
M. S. Meselson, Harvard University
Randall S. Murch, Virginia Polytechnic Institute and State University
Pauline Newman, U.S. Court of Appeals for the Federal Circuit
Stanley A. Plotkin, University of Pennsylvania (emeritus)
Elizabeth Rindskopf Parker, University of the Pacific
R. Paul Schaudies, GenArraytion, Inc.
James M. Tiedje, Michigan State University
Bruce Weir, University of Washington
Although the reviewers listed above provided many constructive comments and suggestions, they were not asked to endorse the conclusions or recom-
mendations, nor did they see the final draft of the report before its release. The review of this report was overseen by Stephen Fienberg, Carnegie Mellon University, and Floyd Bloom, The Scripps Research Institute. Appointed by the National Research Council, they were responsible for making certain that an independent examination of this report was carried out in accordance with institutional procedures and that all review comments were carefully considered. Responsibility for the final content of this report rests entirely with the authoring committee and the institution.
Contents
1.2 Chronology of Events of Fall 2001
1.3 Brief Summary of the FBI’s Scientific Investigation
1.4 Summary of FBI and DOJ Scientific Conclusions
1.6 Issues for Consideration in Reading This Report
1.7 Organization of the Report
2 BIOLOGY AND HISTORY OF BACILLUS ANTHRACIS
2.2 The Biology of B. anthracis
2.3 Clinical Aspects of Anthrax
2.4 B. anthracis as a Biological Weapon
2.6 The Early History of the Ames Strain of B.anthracis
3.2 Science and Scientific Investigation as Part of a Law Enforcement Investigation
3.3 The Federal Coordinated Response and Assignment of Laboratory Work
3.4 Collection and Analysis of Clinical and Environmental Samples and Cross Contamination
3.4.1 Clinical and Epidemiological Samples
3.4.2 Crime Scene Environmental Samples
3.4.3 Samples from an Overseas Site Identified by Intelligence
3.4.4 Letter Material and Cross Contamination
3.5 Committee Findings and Recommendations
4 PHYSICAL AND CHEMICAL ANALYSES
4.2 Spore Preparation and Purification
4.3 Surrogate Preparation and Purification
4.4 Size and Granularity of the Material in the Letters
4.5 Presence of Silicon and Other Elements in the Letter Material
4.5.2 Spatially Resolved Elemental Analysis
4.5.3 Silicon in the Spore Coat
4.5.4 Summary of the Silicon Analysis
4.8 Volatile Organic Compounds
4.9 Determining When the Material Was Produced: Radiocarbon Dating of B. anthracis Samples
4.10.3 The Envelope Measurements
5 MICROBIOLOGICAL AND GENETIC ANALYSES OF MATERIAL IN THE LETTERS
5.2 Identification of the B. anthracis Strain
5.3 Was the B. anthracis in the Letters Genetically Engineered?
5.4 B. subtilis Contamination of the New York Samples
5.5 Identification and Characterization of Colony Morphological Variants in the Evidentiary Material
5.5.1 Why Was the FBI Interested in Colony Morphotypes?
5.5.2 Background Information on Morphotypes
5.5.3 Detection and Characterization of Morphotypes in the Anthrax Letters Samples
5.5.4 Selection Criteria for Genetic Variations Used in Screening
5.5.5 Whole Genome Sequencing of Morphotype Isolates
5.5.6 Development and Application of Assays for the Genotypes
6 COMPARISON OF THE MATERIAL IN THE LETTERS WITH SAMPLES IN THE FBI REPOSITORY
6.2 Creation of the FBI Repository (FBIR)
6.3 Use of the Genetic Assays to Test for the Four Genotypes
6.4 Derivation of RMR-1029 Spores
6.5 Analyses of the Repository Samples and Statistical Interpretation of the Evidence
6.5.1 The FBI’s Statistical Analysis Report
6.5.2 Committee Assessment of the Statistical Analysis Report
6.6 Analyses Based on Resampling RMR-1029 and Interpretation of the Results
INDEX OF DOCUMENTS PROVIDED BY THE FEDERAL BUREAU OF INVESTIGATION
B The Forensics Potential of Stable Isotope Analysis
C Committee Evaluation of Statistical Analysis Report
Tables, Boxes, Figures
TABLES
S-1 FBI and DOJ Conclusions and Committee Comments
1-1 Timeline of Key Events in the Anthrax Mailings Case
3-1 Timeline of Scientific Events in the Anthrax Mailings Investigation
3-2 Analytical Techniques Used on the Evidentiary Material
4-1 Estimated Ranges of Total Number of Spores
4-2 Estimates of Media Volume Required for Spore Preparation
4-3 Methods for Chemical Analysis Referred to in Chapter 4
4-4 Summary of Silicon Measurements in Evidentiary and Surrogate Samples
5-1 Phenotypic Characteristics of the Morphotypes
5-2 B. anthracis Isolates Analyzed by the Institute for Genomic Research (TIGR)
5-3 Further Genetic Characterization of the Morphotype Isolates
5-4 Distribution Among the Anthrax Letters of the Genotypes Selected for Repository Screening
6-4 Observed and Expected (Under Independence) Distribution of Positive Signatures of Four Genotypes
6-6 Results Obtained by Resampling from Flask RMR-1029
C-1 Samples with Positive and “Inconclusive” or “Variant” Assays
C-2 Probabilities of k 4-mutation Samples in Institution F
BOXES
3-1 Bioterrorism Investigations
5-2 The Polymerase Chain Reaction Technique
6-1 Subpoena Protocol for Collection and Submission of Ames Strain Samples
FIGURES
2-1 Worldwide Distribution and Lineages of B. anthracis
3-1 Trajectory and Outcomes of Anthrax Mailings
3-2 New York Post Letter Powder
4-1 SEM of Leahy and New York Post Powders
4-2 Stable Isotope Results 18O and 2H
5-1 B. anthracis Colony Morphotype “A”
5-2 B. anthracis Colony Morphotype “B”