CASE CLOSED … what really happened in the 2001 anthrax attacks?

* April 26, 2012 FBI Quantico Publication: The Detection of Meglumine and Diatrizoate In No Way Pointed To Bruce Ivins As The Perpetrator Or Involved At All; Meglumine and Diatrizoate were both detected in the USAMRIID RMR 1029 sample — but Meglumine and Diatrizoate were NOT detected in the 2001 letter spore evidence

Posted by DXer on May 9, 2012




24 Responses to “* April 26, 2012 FBI Quantico Publication: The Detection of Meglumine and Diatrizoate In No Way Pointed To Bruce Ivins As The Perpetrator Or Involved At All; Meglumine and Diatrizoate were both detected in the USAMRIID RMR 1029 sample — but Meglumine and Diatrizoate were NOT detected in the 2001 letter spore evidence”

  1. DXer said

    The article above is cited in this article by FBI authors.

    Technical Note

    Fatty Acid Profiles for Differentiating Growth Medium Formulations Used to CultureBacillus cereus T-strain Spores†
    • Christopher J. Ehrhardt Ph.D.1,
    • Devonie L. Murphy M.S.2,3,
    • James M. Robertson Ph.D.3,* and
    • Jason D. Bannan Ph.D.4

    Article first published online: 9 APR 2015

    © 2015 American Academy of Forensic Sciences

    Presented in part at the 64th Annual Meeting of the American Academy of Forensic Sciences, February 20–25, 2012, in Atlanta, GA.

    • forensic science;
    • forensic biology;
    • fatty acid methyl esters;
    • spores


    Microbial biomarkers that indicate aspects of an organism’s growth conditions are important targets of forensic research. In this study, we examined fatty acid composition as a signature for the types of complex nutrients in the culturing medium. Bacillus cereus T-strain spores were grown in medium formulations supplemented with one of the following: peptone (meat protein), tryptone (casein protein), soy protein, and brain–heart infusion. Cellular biomass was profiled with fatty acid methyl ester (FAME) analysis. Results showed peptone cultures produced spores enriched in straight-chained lipids. Tryptone cultures produced spores enriched in branched-odd lipids when compared with peptone, soy, and brain–heart formulations. The observed FAME variation was used to construct a set of discriminant functions that could help identify the nutrients in a culturing recipe for an unknown spore sample. Blinded classification tests were most successful for spores grown on media containing peptone and tryptone, showing 88% and 100% correct identification, respectively.

    “The anthrax letter attacks of 2001 were one of the first large-scale incidents of domestic bioterrorism in the United States and resulted in one of the most complex investigations undertaken by the Federal Bureau of Investigation.”

    The authors include members of the FBI’s Counterterrorism and Forensic Science Research Unit and an FBI Biological Program Science Advisor.

  2. DXer said

    IVINS first became aware of heat shocking back in the Gulf War. The Division Chief called IVINS in his office to discuss a large NHP [nonhuman primate] study. The Division Chief did not want the spores Renograffin purified because that was not done in the old days. Spores used to be heat shocked at…..

    This temperature was chosen because counts do not decrease at this temperature and refractile spores are not killed. Spores that are used from intramuscular (IM) or aerosol challenges are heat shocked. All spores are heat shocked in building 1412. IVINS does not know if anyone has studied virulence with respect to heat shock. Howver, _________________ did some studies on heat shocked material versus non-heat shocked material.

    Ivins 302, 09/08/2004, page 10

  3. DXer said

    Was the trace detection of meglumine and diatrizoate in samples of virulent Ames focused on in 2002 and 2003?

    Fusion of laboratory and textual data for investigative bioforensics
    BJ Webb-Robertson, C Corley, LA McCue… – Forensic science …, 2013 – Elsevier


    Chemical and biological forensic programs focus on the identification of a threat and acquisition of laboratory measurements to determine how a threat agent may have been produced. However, to generate investigative leads, it might also be useful to identify institutions where the same agent has been produced by the same or a very similar process, since the producer of the agent may have learned methods at a university or similar institution. We have developed a Bayesian network framework that fuses hard and soft data sources to assign probability to production practices. It combines the results of laboratory measurements with an automatic text reader to scan scientific literature and rank institutions that had published papers on the agent of interest in order of the probability that the institution has the capability to generate the sample of interest based on laboratory data. We demonstrate the Bayesian network on an example case from microbial forensics, predicting the methods used to produce Bacillus anthracis spores based on mass spectrometric measurements and identifying institutions that have a history of growing Bacillus spores using the same or highly similar methods. We illustrate that the network model can assign a higher posterior probability than expected by random chance to appropriate institutions when trained using only a small set of manually analyzed documents. This is the first example of an automated methodology to integrate experimental and textual data for the purpose of investigative forensics.

    Background –

    FBI Study Raises More Questions in Bruce Ivins Anthrax Case
    MAY 29, 2012

    An article published last week by the Center for Biosecurity at the University of Pittsburgh Medical Center (UPMC) describes how a recent study adds more questions to the controversial investigation of USAMRIID scientist Dr. Bruce Ivins as the perpetrator of the 2001 anthrax mailing attacks in the United States.

    The initial FBI investigation linked spores found in the anthrax mailings to material contained in a flask labeled “RMR-1029” that was under Dr. Ivins’ control. Lab records later determined that the RMR-1029 material had been purified using a product containing two specific ingredients: meglumine and diatrizoate.

    To determine if spore material from the letters also contained these ingredients, researchers from the CBRN Sciences Unit of the FBI developed novel methods to detect trace elements of meglumine and diatrizoate in dried anthrax spores.

    Investigators confirmed the presence of the ingredients in the USAMRIID flask. However, utilizing the same methods they found no evidence of the ingredients in the spore samples from the letters used in the attacks.

    The research study titled “Trace Detection of Meglumine and Diatrizoate from Bacillus Spore Samples Using Liquid Chromatography/Mass Spectrometry” was published last month in the Journal of Forensic Scientists.

    – See more at:

    Comment: An automated reader is not necessary to adopt a Bayesian approach. There just needs to be someone at the FBI’s hazardous materials unit in 2002 and 2003 doing the necessary reading and analysis using boolean search capability.

    Was the trace detection of meglumine and diatrizoate in samples of virulent Ames focused on in 2002 and 2003?

    Separately, it was the job of the hazardous materials unit to read the lab notebooks, protocols and emails relating to Dr. Ivins’ experiment with the 52 rabbits and formaldehyde. It seems that some scientist relied instead on the prosecutor’s Cliff notes version and investigative spin.

    DXer summarizes the documentary evidence relating to Dr. Ivins work with rabbits (nowhere mentioned by the DOJ) which demolishes the FBI’s claim that Dr. Ivins had no reason to be in the lab

    Posted by Lew Weinstein on July 3, 2012

  4. DXer said

    Last year on PRO MED, there was discussion of this issue.

    Published Date: 2012-05-13 17:19:51
    Subject: PRO/AH> Anthrax, human, 2001 – USA (02): chemical RFI
    Archive Number: 20120513.1131856
    A ProMED-mail post
    ProMED-mail is a program of the
    International Society for Infectious Diseases

    Date: May 11, 2012
    From: Dennis H. Grant, MD

    I have been following the reports on Promed about the anthrax attack, as well as reviewing the FBI report, and recent books written about the events, and have pieced together some interesting, hopefully relevant thoughts.

    The recent posting about the lack of meglumine and diatrizoate on the evidentiary spores matches a statement from the DOJ/FBI report (1) that indicates that there were no traces of renografin on the mailing material, a substance that apparently contains meglumine and diatrizoate. According to the recent post, the RMR-1029 flask contained spores that had been purified using materials containing these substances, indicating that direct use of these spores was not involved.

    Basically this inconsistency runs against the FBI finding that the genetic analysis of the evidentiary spores linked RMR-1029 flash to the attack. The quote from the FBI report indicates that water purification could have been used to purify the evidentiary spores, accounting for the lack of renografin substances. The question becomes, then, whether water washing of the spores from RMR-1029 could have removed traces of meglumine or diatrizoate from the spores, still linking Dr. Ivins to the attack.

    I could not find in the FBI report something to say that washing spores from RMR-1029 could have removed the renografin (meglumine or diatrizoate). Would anyone have the answer to this? Otherwise, Dr. Ivins would have had to obtain fresh spores, equal genetically to those found by the FBI, and then water washed them for purification purposes. I wonder about the probability of this.

    Now, having read the 2 books on the investigation, “American Anthrax,” by Jeanne Guillemin, and “The Mirage Man,” by David Willman, I am of the impression that the FBI used the Reid method of interrogation on Dr. Ivins, i.e., one which basically involves 1st interviews, followed by accusatory interrogation once they have felt they have found the perpetrator of a crime, in order to extract a confession. In reading the accounts, I often wondered why an indictment was not pursued considering the circumstantial evidence. Perhaps the reason was that the inconsistency was known by authorities, could not adequately be explained, and thus would have had to be revealed to the defense as exculpatory evidence.

    My own thought is that the case is not yet closed.

    Footnote: (1) “An expert microbiologist from Dugway stated that these hours (comment: a period of 69 hours that occurred between the 1st and 2nd mailings, a time assumed to be used for further purification of spores, and a time for which Dr. Ivins could not account to any degree) were consistent with someone preparing the anthrax letters. He specifically noted that the spore-washing process would take some time, especially if the mailer were not using a density gradient to clean the spores. This is significant because there are no traces of renografin on the mailing material, which means that the spores could have simply been water-purified, as opposed to this other measure. The expert noted that with respect to the material in the 1st mailings, he would have been “embarrassed to send that out” because it was so granular – further support for the notion that the additional time expended in preparing the 2nd round of materials may have been due to further washing/purifying of the spores” (From United States Department of Justice, Ameritrax Investigation Summary, February 10, 2010, page 32).

    — Dennis H. Grant, MD (Retired from federal employment – Army, IHS, VA), specialty psychiatry 4211 E Paradise Dr. Phoenix, Arizona 85028

    [Dennis raises the question whether the renografin (meglumine or diatrizoate) could have been washed off during washing the spores. A fair question and if there any members with sound expert knowledge on this subject, we would be glad to hear from them.

    If the renografin chemicals cannot be removed by washing this would put the letter spores’ origin immediately away from the RMR-1029 flask, even if cultured from a common source or from material shared taken from the flask. I would remind members that the letter contents had a _B. subtilis_ contaminant, which was not found in the contents of the RMR-1029 flask. While the FBI dismissed the importance of this contaminant Bacillus it has the potential of being an institutional fingerprint. – Mod MHJ]

  5. DXer said

    This article establishes that the spores did NOT come directly from flask RMR-1029.

    • DXer said

      This analysis is sufficient, everyone agrees, to answer one question: Were the letters taken directly from RMR-1029 and used without further purification? The answer is no. The presence of diatrizoate and meglumine in RMR-1029 samples and their absence in the letter samples is consistent with the idea that the letter spores were not derived directly from RMR-1029 The resulting spores were either not purified using diatrizoate and meglumine or they were washed extensively following purification, lowering the diatrizoate and meglumine to levels below the assay’s limits of detection. A large amount of virulent Ames made by Patricia Fellows intended to replenish Flask 1029 but reportedly never put in Flask 1029 is missing. See Amerithrax Investigative Summary. The DOJ has shredded her civil deposition.

      • DXer said

        Relatedly, this powdered sample of virulent anthrax also went missing.

        GAO: Did Patricia Fellows Ever Find the Missing “National Security” Sample That Dr. Ivins Was (Apparently Falsely) Told Was From Iraq Before Moving On To SRI That Summer? Was There An Emailed Response(s) To Dr. Ivins’ Question? Her Deposition Should Not Be Shredded. [but it was]

        Posted by Lew Weinstein on December 14, 2011

      • DXer said

        The FBI scientists in their important 2012 article cite this article:

        Residual Agar Determination in Bacterial Spores by Electrospray Ionization Mass Spectrometry

        • Abstract

        Karen L. Wahl *, Heather A. Colburn , David S. Wunschel , Catherine E. Petersen , Kristin H. Jarmanand Nancy B. Valentine
        Pacific Northwest National laboratory, P.O. Box 999, MS P7-50, Richland, Washington 99352
        Anal. Chem., 2010, 82 (4), pp 1200–1206
        Publication Date (Web): January 14, 2010
        Copyright © 2010 American Chemical Society
        * To whom correspondence should be addressed. Phone: 509-371-6853. Fax: 509-371-6679.

        Presented here is an analytical method to detect residual agar from a bacterial spore sample as an indication of culturing on an agar plate. This method is based on the resolubilization of agar polysaccharide from a bacterial spore sample, enzymatic digestion, followed by electrospray ionization tandem mass spectrometry (ESI-MSn) analysis for detection of a specific agar fragment ion. A range of Bacillus species and strains were selected to demonstrate the effectiveness of this approach. The characteristic agar fragment ion was detected in the spores grown on agar that were washed from 1 to 5 times, irradiated or nonirradiated, and not in the spores grown in broth. A sample containing approximately 108spores is currently needed for confident detection of residual agar from culture on agar plates in the presence of bacterial spores with a limit of detection of approximately 1 ppm agar spiked into a broth-grown spore sample. The results of a proficiency test with 42 blinded samples are presented demonstrating the utility of this method with no false positives and only three false negatives for samples that were below the detection level of the method as documented.

        Citing articles:

        • Catherine Swider, Kelly Maguire, Michael Rickenbach, Madeline Montgomery, Matthew J. Ducote, Craig A. MarhefkaJournal of Forensic Sciences 2012, 57 (4), 923-931 [CrossRef]
        • David S. Wunschel, Karen L. Wahl, Angela M. Melville, Christina M. Sorensen, Heather A. Colburn, Nancy B. Valentine, Casey L. StamperTalanta 2011, 85 (5), 2352-2360 [CrossRef]
        • Heather A. Colburn, David S. Wunschel, Kathryn C. Antolick, Angela M. Melville, Nancy B. ValentineJournal of Microbiological Methods 2011, 85 (3), 183-189 [CrossRef]
        • Karen L. Wahl, David S. Wunschel, Brian H. Clowers. 2011. Proteomics Development and Application for Bioforensics. , 449-460. [CrossRef]
        • Journal of Mass Spectrometry 2010, 45 (6), 686-697 [CrossRef]

  6. DXer said

    Evidence Based on Spore Purification Compounds

    During the Amerithrax investigation, it was discovered that some laboratories that possessed the Ames strain were purifying the spores using a product that contained meglumine and diatrizoate. From lab records, it was determined that the spores contained in RMR-1029 were similarly purified using these compounds. This information gave rise to the question of whether the attack materials were taken directly from RMR-1029. To answer this question, the FBI developed a detection method that employed liquid chromatography and mass spectrometry to determine the presence of these compounds.1 As expected, the FBI investigators did find that anthrax spores taken directly from RMR-1029 contained both meglumine and diatrizoate. However, no evidence of these compounds was found when the spores from the anthrax letters were analyzed using the same method.1

    Source of Attack Material?

    The major implication of this study, as the authors note, is “that the evidentiary spore material was not diverted directly from RMR-1029.”1 This fact means that if the anthrax spores used in the attack were taken from RMR-1029, their preparation would have required extra steps prior to mailing. That type of purification would have required specialized machinery and likely would have left traces of the material on machinery. No such material was found, though, and in a recently settled civil case in Florida, the U.S. Department of Justice acknowledged that the specialized machinery was not available at USAMRIID.2

  7. DXer said

    Published Date: 2012-05-10 18:04:59
    Subject: PRO/AH/EDR> Anthrax, human, 2001 – USA: chemical evidence
    Archive Number: 20120510.1129210
    A ProMED-mail post
    ProMED-mail is a program of the
    International Society for Infectious Diseases

    Date: Thu 10 May 2012

    Source: Journal of Forensic Sciences [edited] [subscription required for full text]

    [Ref: Swider C, Maguire K, Rickenbach M, et al: Trace detection of meglumine and diatrizoate from _Bacillus_ spore samples using liquid chromatography/mass spectrometry. J Forensic Sci. 2012 Apr 26. doi: 10.1111/j.1556-4029.2012.02128.x. [Epub ahead of print]


    Federal Bureau of Investigation, Laboratory Division, Chemical Biological Radiological and Nuclear Sciences Unit, 2001 Investigation Parkway, Quantico, VA 22135, USA



    Following the 11 Sep 2001 terrorist attacks, letters containing _Bacillus anthracis_ were distributed through the United States postal system killing 5 people. A complex forensic investigation commenced to identify the perpetrator of these mailings. A novel liquid chromatography/mass spectrometry protocol for the qualitative detection of trace levels of meglumine and diatrizoate in dried spore preparations of _B. anthracis_ was developed. Meglumine and diatrizoate are components of radiographic imaging products that have been used to purify bacterial spores. 2 separate chromatographic assays using multiple mass spectrometric analyses were developed for the detection of meglumine and diatrizoate. The assays achieved limits of detection for meglumine and diatrizoate of 1.00 and 10.0 ng/mL, respectively. _Bacillus cereus_ T strain spores were effectively used as a surrogate for _B. anthracis_ spores during method development and validation. This protocol was successfully applied to limited evidentiary _B. anthracis_ spore material, providing probative information to the investigators.



    In the weeks following the 11 Sep 2001 terrorist attacks on the World Trade Center and the Pentagon, 4 letters containing _Bacillus anthracis_ spores were collected. These letters were addressed to 2 media outlets in New York City and to 2 members of the United States Senate in Washington, DC. As a result of the distribution of these letters through the United States Postal System, 5 victims died and at least 17 victims demonstrated symptoms of inhalational or cutaneous anthrax.

    The investigation to determine the individual(s) responsible for the most disruptive terrorist attack on the United States involving the use of a biological agent was conducted by the Federal Bureau of Investigation (FBI) and the United States Postal Inspection Service (USPIS). Because of unprecedented challenges, this was among the most complex investigations the FBI or the USPIS had ever conducted. The investigation team, known as the Amerithrax Task Force, worked with subject matter experts and the scientific community to develop novel analytical/forensic assays to leverage all possible information from the evidentiary _B. anthracis_ spore materials.

    The _B. anthracis_ spores recovered from the letters were determined to be of the Ames strain and were of a high degree of purity. While the spores recovered from the mailings to media outlets in New York City were characteristically different (for example, off-white in color, more granular, some cellular debris, and growth media components present) from the spores sent to Washington, DC., both had high colony-forming units (CFU) per gram of material, on the order of 1011 CFU/g, indicating that both were high-quality spore preparations. Spores of such purity are often used in conducting aerosol challenges to minimize the incidence of nebulizer obstruction by cellular debris or growth media components during an experiment.

    The investigation determined that some laboratories conducting _B. anthracis_ research with the Ames strain were purifying spores using a density gradient of RenoCal-76(R) or similar products. Meglumine diatrizoate and sodium diatrizoate are the primary constituents in RenoCal-76(R), Hypaque-76(R), and Renografin-60(R), which are commercially available radiographic imaging products. In addition, meglumine diatrizoate, meglumine, and sodium diatrizoate are readily available from commercial chemical suppliers. The literature reports, as early as 1966, the use of products containing meglumine diatrizoate in spore purification.

    The Ames _B. anthracis_ used in the New York City and Washington, DC mailings had a number of identified morphological variants, which were isolated and their complete genomes sequenced. The sequences of these variants were compared to the wild-type Ames _B. anthracis_, and a number of genetic differences were identified. Assays were used to screen over 1000 samples of Ames _B. anthracis_ collected from research institutions within the United States and internationally. Of the samples screened, all samples positive for all of the genetic markers were determined to originate from a common source of spores, known as RMR-1029. The RMR-1029 spores were known, from laboratory records, to have been purified using a density gradient of RenoCal-76(R). Some investigative questions became: “Were the evidentiary spores from the mailings directly diverted from RMR-1029? Could an analytical method identify residual RenoCal-76® in a spore preparation known to be purified using RenoCal-76(R)?”

    This paper describes the development, validation, and application of a novel, highly sensitive protocol using liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization (ESI) to detect trace amounts of meglumine and/or diatrizoate, components of RenoCal-76(R), in a single spore sample preparation. This analytical capability was applied to limited evidentiary spore material and RMR-1029 to provide probative information about a possible production method of the evidentiary samples. During the investigation, it was determined that the number of researchers who used gradients of RenoCal-76(R), or similar products, to purify spores and who had access to the Ames strain of _B. anthracis_ was limited. Therefore, if meglumine and diatrizoate were identified in the _B. anthracis_ spores used in the mailings, the number of potential sources for the spore material could be significantly reduced.



    A sensitive and selective analytical protocol has been developed for the detection of meglumine and diatrizoate in samples of _Bacillus_ spores. A tiered approach of capturing chromatographic separation, full-scan MS, MS2, and MS4 data was developed for both meglumine and diatrizoate. System carryover concerns with meglumine were resolved by changing the stationary and mobile phases. The method validation demonstrated both sensitivity and selectivity by obtaining detection limits of meglumine and diatrizoate at concentrations ranging from 1.00 to 10.0 ng/mL. Maximizing the data that could be derived from the analysis of a few milligrams of evidentiary material was paramount to the FBI. The application of this novel method proved to be a valuable tool during the investigation. As the genetic data that linked the _B. anthracis_ spore material from the mailings to RMR-1029 was being compiled, investigators were uncertain whether an aliquot of RMR-1029 was used directly. The absence of meglumine and diatrizoate on the evidentiary material, using the protocol described herein and when taken together with other forensic examinations, was supportive to the investigation in indicating that the evidentiary spore material was not diverted directly from RMR-1029.

    Communicated by:


    [The key sentence is the last: “The absence of meglumine and diatrizoate on the evidentiary material … was supportive to the investigation in indicating that the evidentiary spore material [in the letters] was not diverted directly from RMR-1029.” So Bruce Ivins could not have brewed up these spores working after hours as proposed by the FBI. It had to have been done elsewhere in an institute that did not employ “RenoCal-76(R) or similar products to purify spores.”

    It is hard to understand why it has taken so long for this information to be published, more than 10 years since the events of October 2001. One can think of various scenarios but Swider and her colleagues, and their superiors, are to be congratulated on their institutional courage as there must have been pressures to not do so. – Mod.MHJ]

  8. DXer said

    Dr. Adams should be asked his understanding of this 2012 peer-reviewed article by the scientists from the FBI Laboratory Division.

  9. DXer said

    FBI Study Raises More Questions in Bruce Ivins Anthrax Case

  10. DXer said

    Computer forensics was a big part of the Amerithrax case. The GAO should review the FBI’s use of computer forensics. As Attorney Kemp explained, none of that evidence implicated Dr. Ivins. However, GAO review of the material might provide important insights. Indeed, finding the missing laptop that Dr. Ivins suggested Pat Fellows had last in 2002 could be critical evidence relating to the work that was done in the B3 relating to the rabbits in late September 2001/early October 2001.

    The FBI takes advantage of the fact that people who traffic in pornography with strangers do not understand the FBI’s capabilities. Finding child pornography on a pornographer’s computer is powerful evidence, without more, of a felony.

    ABC 20/20 this year discussed a Special Agent’s Affidavit in a national security matter. The piece notes: “”The analysis could directly implicate or eliminate the suspect based on the information recovered, or serve as corroboration or contradiction to a suspect or witnesses statement,” said FBI Supervisory Special Agent Sean O’Brien, director of the Rocky Mountain Regional Computer Forensics Lab in Centennial, Colorado.”

    Finding the missing Apple laptop that Patricia Fellows last had would provide contemporaneous documentation of the work with the rabbits — the research for which the FBI withheld ALL hard copies. Electronic documentation tends to be more extensive than hard copies because much that is created electronically is never printed.

    If the Apple laptop were located, the GAO’s herculean task would be considerably simplified.

    Jan. 30, 2012

    In an affidavit, FBI Special Agent Donald Hale noted that Muhtorov communicated with associates using two email addresses, an Android Blackberry smart phone and a Sony Vaio laptop computer that Hale suggested could yield a bounty of information.

    When “Delete” Does Not Mean Delete

    “Computer files or remnants of such files can be recovered months or even years after they have been downloaded onto a storage medium, deleted, or viewed via the Internet,” Hale wrote in the affidavit. “Even when files have been deleted, they can be recovered months or years later using forensic tools.”

    Hale explained that when a person deletes a file on a computer, the data doesn’t actually disappear, but remains on the hard drive until it gets overwritten by new data. The computer’s operating system may also keep records of deleted files in something called a “swap” or “recovery” file.

    A computer’s internal hard drive can keep records of how it was used, who used it, and when, Hale wrote. This digital evidence can point to information that once lived on a hard drive or memory stick, but was later altered or deleted. For example, agents might even be able to see where an incriminating paragraph was erased from a word processing document.

    “Computer users typically do not erase or delete this evidence, because special software is typically required for that task,” agent Hale wrote.

    The trail doesn’t end there. Web browsers, email and chat programs can reveal online nicknames and passwords. The computer can also tell investigators when a memory stick or external hard drive was connected, and how and in what sequence files were created.

    Analyzing all that electronic evidence, Hale wrote, takes “considerable time.”

    That work gets done at one of 16 computer forensics laboratories around the country run by the FBI, in partnership with 130 state and local law enforcement agencies. The first Regional Computer Forensics Laboratory, as they are officially called, was established in San Diego in 1999.

    Agents who first obtain court approved search warrants can scour cell phones, cameras, GPS units, tablet computers and more for information that can make or break an investigation.

    Digital Detectives

    “The analysis could directly implicate or eliminate the suspect based on the information recovered, or serve as corroboration or contradiction to a suspect or witnesses statement,” said FBI Supervisory Special Agent Sean O’Brien, director of the Rocky Mountain Regional Computer Forensics Lab in Centennial, Colorado.

    In the 2010 fiscal year, the regional laboratories conducted 6,564 examinations of everything from hard drives and cell phones to floppy disks and VHS videotapes. During that time examiners combed through 3,086 terabytes of data. (For comparison, just one terabyte is equal to about 1,000 gigabytes.)

    The digital deluge can be overwhelming.

    “The sheer volume of information investigators request to be analyzed exceeds the capacity of forensic examiners available to analyze the data in the laboratory,” O’Brien told ABC News.

  11. DXer said

    I have posted excerpts and Martin Hugh-Jones has posted excerpts on Pro-Med.

    It is public domain as a matter of law — written by government scientists funded by taxpayers.

    I would be glad to provide anyone who likes a copy of the full-text.

  12. DXer said

    “This paper describes the development, validation, and application of a novel, highly sensitive protocol using liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization (ESI) to detect trace amounts of meglumine and/or diatrizoate, components of RenoCal-76®, in a single spore sample preparation. This analytical capability was applied to limited evidentiary spore material and RMR-1029 to provide probative information about a possible production method of the evidentiary samples. During the investigation, it was determined that the number of researchers who used gradients of RenoCal-76®, or similar products, to purify spores and who had access to the Ames strain of B. anthracis was limited. Therefore, if meglumine and diatrizoate were identified in the B. anthracis spores used in the mailings, the number of potential sources for the spore material could be significantly reduced.”

    It was not found in the mailed anthrax. Dr. Ivins used it. Thus, it tends to be exculpatory of Dr. Ivins.

    • DXer said

      The authors note:

      “As previously mentioned, the investigation was complex and many forensic analyses were conducted. To further minimize consumption of evidence, investigators requested this assay be developed using a minimal amount of spore material. As a result, the method was developed and validated using c. 2 mg of irradiated dry spore material. It is appropriate to highlight the difficulty encountered in obtaining an accurate weight of such a small amount of dried spore material owing to the electrostatic properties. A zerostat antistatic instrument and antistatic vinyl gloves were used during the manipulations of any dried spore material. Weighing small quantities of spore material proved to be challenging and may have been problematic had a quantitative assay been pursued.”

    • DXer said

      The authors emphasize:

      “An analysis of RMR-1029 using the validated, multiple-tiered approach described herein resulted in a positive response in all assays for meglumine and a positive response for LC/MS2 and LC/MS4 for diatrizoate. These data confirmed that residual meglumine and diatrizoate were identified on the RMR-1029 spores. When the meglumine and diatrizoate assays were applied to the evidentiary spore material, neither meglumine nor diatrizoate was detected within the LODs.”

  13. DXer said

    Heat shocking in the old days was done instead of using renografin — all heat shocking was done in Building 1412.

    The FBI Should Produce To GAO All Of The Research, Unpublished Or Not, By Dr. Bannan And His Colleagues On Heat Shocking As A Substitute For Use Of Renografin In Purification, To Include Whether Heat Shocking Was Used In Connection With The Fall 2001 Mailings, Whether It Was Used For Intramuscular And Aerosol Challenges Done In USAMRIID Building 1412, And Whether Heat Shocking Was Studied In Connection With Virulence Studies.

    Posted by Lew Weinstein on April 23, 2012

  14. DXer said

    In the filmed question and answer, the FBI’s leading anthrax scientist, John Ezzell, explained to me that the fact that he used renografin for purification confirms the dried powder he made from Flask 1029 was not used in the anthrax mailings.

    The same logic, by the way, applies to what was in Flask 1029.

    By way of some additional background …

    RenoCal-76 = Diatrizoate Meglumine

    and so it can be inferred that RenoCal was not used to purify what was mailed.

    Here are some emails on the general subject.


    Sent: Tuesday, May 08, 2001 2:32 PM
    To: ‘Ivins, Bruce E Dr USAMRIID’
    Subject: RE: Spore information

    BRUCE: Please educate me a little – many of the older papers discuss
    “purification” of the spores with Renografin-76. Is this the same material
    as Hypaque-76 or can it be used as the same? If these are not
    interchangeable, do you know of a current source for the Renografin-76?
    Thank for your help

    From: Ivins, Bruce E Dr USAMRIID
    To: ”
    Subject: RE: Spore information
    Date: Tuesday, May 08, 2001 2:46:35 PM
    – Yes, we used to use Renografin-76. The company stopped making it and started making Renocal-
    76 instead, so we began using that. Then Renocal-76 was taken off the market, so we began using
    Hypaque-76, which performs just the same as Renografin-76 and Renocal-76 as far as spore
    purification goes.
    – Bruce

    • DXer said

      The Hypague-76 referenced in the discussion between Bruce and Battelle is another brand of diatrizoate meglumine and diatrizoate sodium.

      Proposed Conclusions:

      1. Ames taken directly from Flask 1029 was not used.

      2. Ames purified using the agents used by Bruce Ivins was not used.

      3. The Ames from Flask 1029 made into a dried powder by the FBI scientist who threw out Dr. Ivins February 2002 sample was not used.

      4. The person who processed the anthrax did not use renografin or Renocal-76 or Hypaque-76.

      • DXer said

        Rauf Ahmad, who was attending the Porton Down conferences at the request of Ayman Zawahiri, said he had learned some processing tricks from an attendee at one of the Porton Down conferences. He said he had made some internet connections. Who was he sharing processing tricks with? What computer was used for correspondence? Given that Rauf Ahmad was captured, as I recall, in 2001, he was long available to the FBI for questioning. Even though they took a year before questioning Yazid Sufaat, did the FBI at least interview Rauf Ahmad right away given the billions given to Pakistan in aid? Rauf is as chatty as Yazid but unlike Yazid, Rauf is not particularly religious. He is mainly motivated by money.

    • DXer said

      Gamma Irradiation Can Be Used To Inactivate Bacillus anthracis Spores without Compromising the Sensitivity of Diagnostic Assays▿
      Leslie A. Dauphin, Bruce R. Newton, Max V. Rasmussen†, Richard F. Meyer‡ and Michael D. Bowen*
      + Author Affiliations

      Bioterrorism Rapid Response and Advanced Technology (BRRAT) Laboratory, Division of Bioterrorism Preparedness and Response (DBPR), National Center for Preparedness, Detection, and Control of Infectious Diseases (NCPDCID), Centers for Disease Control and Prevention (CDC), Atlanta, Georgia 30333

      The use of Bacillus anthracis as a biological weapon in 2001 heightened awareness of the need for validated methods for the inactivation of B. anthracis spores. This study determined the gamma irradiation dose for inactivating virulent B. anthracis spores in suspension and its effects on real-time PCR and antigen detection assays. Strains representing eight genetic groups of B. anthracis were exposed to gamma radiation, and it was found that subjecting spores at a concentration of 107 CFU/ml to a dose of 2.5 × 106 rads resulted in a 6-log-unit reduction of spore viability. TaqMan real-time PCR analysis of untreated versus irradiated Ames strain (K1694) spores showed that treatment significantly enhanced the detection of B. anthracis chromosomal DNA targets but had no significant effect on the ability to detect targets on the pXO1 and pXO2 plasmids of B. anthracis. When analyzed by an enzyme-linked immunosorbent assay (ELISA), irradiation affected the detection of B. anthracis spores in a direct ELISA but had no effect on the limit of detection in a sandwich ELISA. The results of this study showed that gamma irradiation-inactivated spores can be tested by real-time PCR or sandwich ELISA without decreasing the sensitivity of either type of assay. Furthermore, the results suggest that clinical and public health laboratories which test specimens for B. anthracis could potentially incorporate gamma irradiation into sample processing protocols without compromising the sensitivity of the B. anthracis assays.


      Received 7 March 2008.
      Accepted 20 May 2008.
      ↵*Corresponding author. Mailing address: BRRAT Laboratory, DBPR, NCPDCID, CDC, Mail Stop G-42, 1600 Clifton Road, Atlanta, GA 30333. Phone: (404) 639-4922. Fax: (404) 639-4234. E-mail: MKB6@CDC.GOV
      ↵† Present address: Biosciences Defense Division, Lawrence Livermore National Laboratory, 7000 East Ave., L-452, Livermore, CA 94551.
      ↵‡ Present address: The Tauri Group, 675 N. Washington St., Suite 220, Alexandria, VA 22314.
      ↵▿ Published ahead of print on 30 May 2008.
      American Society for Microbiology

Leave a Reply

Fill in your details below or click an icon to log in: Logo

You are commenting using your account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s

%d bloggers like this: