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Generic Integrated Forensic Toolbox for CBRN incidents

Final Report Summary - GIFT CBRN (Generic Integrated Forensic Toolbox for CBRN incidents)

Executive Summary:
Executive summary
Forensics research plays an important role in solving crime and maintaining secure societies. Novel methods for CBRN forensics and training would strongly enhance these capabilities. Within the GIFT project partners developed and provided a forensic toolbox (mobile) focusing on procedures, practices and guidelines for common CBRN forensic measurements and handling instructions on a European level and storing data form the crime scene investigation, such that results can be used during legal prosecution to provide solid and court-proof forensic evidence in and after CBRN incidents.
Practices were developed for sampling, preservation, shipping and storage, analysis, laboratory equipment and recording in the context of criminal events. Guidelines and procedures included issues like Good Laboratory Practice (GLP), Quality Assurance (QA), Quality Control (QC) and Standard Operating Procedures (SOPs).
The developed procedures, practices and guidelines, projects showed adequate
attention to aspects of usability, societal acceptance and economic and legal viability, through appropriate research, experimentation or demonstration at the GIFT developed scenario’s and exercises. The project liaised with EDEN, TOXITRIAGE and ROCSAFE
in relation to the forensic area. The developed new technologies for sampling, analysing evaluating, interpreting and recording forensic evidence with a view to achieve court-proof results were tested on several stage in the laboratory and at the exercises.
The CBRN forensic procedures demonstrated to be useful as guidelines on how to act in CBRN incidents, in particular how to sample, analyse, evaluate, interpret and record forensic evidence and achieve court-proof results. Some of these guidelines have been used in the DG DEVCO Centers of Excellence project P-57 “Strengthening CBRN crime scene investigation in the Balkan and Black Sea region”.
Testing and validation on the field with relevant end users have illustrated the EU added value of such an initiative. It also included key qualitative and quantitative indicators to measure progress or results achieved during the project compared to the state of the art.
This research related particularly to the goals outlined in the EU CBRN action plan.

Project Context and Objectives:
GIFT, the European CBRN forensics approach
In the last two decades Europe is facing possible CBRN threats caused by industrial incidents but also due to the rising criminal and terrorist activities. On both on national and European level enormous efforts have been developed to protect the European population but also to mitigate these incidents. On national level several EU member states have developed civil response to investigate the CBRN incidents in order to reconstruct he event, relate the CBRN agents to a possible source and prosecute and convict the perpetrators. The forensic methods used to investigate CBRN incidents were derived from the existing classical forensic methods and do need further improvement for use in any CBRN forensic investigation.
To further develop CBRN forensics methods the 21 partners of the GIFT consortium developed a proposal within the frame of FP7 Topic SEC-2013.1.5-1 European toolbox, focusing on procedures, practices and guideline for CBRN forensic aspects – Capability project. The proposal of the GIFT project, Generic Integrated Forensic Toolbox to investigate CBRN incidents, has been focused on procedures, methods, instruments and guidelines for forensic investigators at the CBRN crime scene, methods to decontaminate exhibits so that traditional methods can be applied safely, develop methods that can be applied in containment and laboratory methods to identify the CBRN agents and characterize the agents to relate them to a possible source.
To further support the GIFT developments the digital GIFT toolbox where all GIFT developments will come together to make them accessible for forensic investigators and the decision makers. Within the Toolbox also modalities to store data derived by the developed equipment and a graphic user interface to visualize the crime scene, the location of the seized exhibits and the measurements of the hazardous materials.
For crime scene investigation equipment and methods have been developed to identify and locate e.g. α-and γ-radiating sources by a camera, analysis methods for nerve agents by lab-on-a-chips (μTAS), volatile chemicals by HRIMS and the use of swaps as a collection methods for chemical warfare agents. Also a portable PCR instrument to be used near the crime scene to identify biological agents. To support the crime scene investigators in their work procedures for sampling, packaging, transport and chain of custody requirements have been developed.
In order to investigate traditional forensic traces, such as human DNA, latent fingerprints and digital data carriers, decontamination methods have been developed with mVHP and VDC (Vacuum Chamber Decontamination) to avoid alteration or destruction of forensic traces. The developed decontamination methods have tested with the available accredited methods for DNA analysis, visualizing latent fingerprints and retrieving data from digital data carriers. In the investigations the developed VDC decontamination method showed the better results in both decontamination efficiency and not destroying forensic traces. In case when it is impossible to decontaminate the exhibits the accredited forensic methods have been modified and tested for use in a containment such as e.g. a glove box.
CBRN forensics, as the forensic investigation on the different C, B, R and N agents is called, has been focused on the identification of the agents and characterizing them so that the materials can be related to a possible source, which is very worthy information for law enforcement. Developments have been done on characterizing toxins in a specimen of mushrooms. With the developed method we could distinguish mushrooms of the same specimen coming from different locations. For attribution of biological agents a statistical method has been developed and tested to analyze the retrieved DNA profiles. In addition methods have been developed for attribution of chemical warfare agents.
As we do see implementation of the GIFT toolbox in the EU member states is of paramount importance, an education and training curriculum has been developed to facilitate the implementation by law enforcement agencies and forensic organizations.
Because CBRN forensics methods can be high end technologies (advanced) and implementing CBRN methods in one of the EU member states the GIFT road map has been developed as tool for EU member states how to select and implement CBRN forensics tools in their countries. For the implementation of the GIFT tools member states can start with implementing core capabilities that are needed for the CBRN forensics response in their country. For the advanced capabilities it is advised for member states to internally discus the need of implementing these capabilities as there are EU-organizations like JRC, national universities or specialized laboratories in neighboring states to assist in the investigations. In the latter case bilateral agreements can be set up so that a country do have access to the investigative capabilities in other countries.
During the GIFT project we have not only identified law enforcement organizations and forensic investigator for which the GIFT toolbox will be very helpful, but also defense organizations within the EU member states, the EDA, EUROPOL, INTERPOL, the IAEA and the OPCW showed their interest in the GIFT developments
For a GIFT 2 project in the future it was recognized to further develop specialized detectors supporting crime scene investing, enhance the traditional forensic methods and continue developing characterization methods for the CBRN agents so that attribution of the materials can be done unambiguously.
Of great interest for the CBRN forensic workfield is the use of drones and robotics in the crime scene investigation. A development that is covered at the moment with the Horizon 2020 project ROCSAFE.

Project Results:
Main S&T results
1. WP3, Tools on Scene
The objectives of WP3 are to develop procedures, guidelines and technology to enable faster, cheaper, accurate and reliable crime scene investigation of CBRN incidents.
Science and Technology results of WP3 are:
T3.1: Registration of crime scene with 3-D imaging
From the results of this deliverable, a number of general recommendations for preparations can be given:

A simple and relatively cheap, but also necessary, preparation is to organize regular tests and field exercises with new, and cheap affordable, wireless working drones, PTZ-camera’s and sensors. Consider if they should be part of the standard CSI-equipment. Avoid circumstances that a lot of time is needed to get acquainted with these devices and to make them work.

A necessary preparation that requires investments in information technology and cooperation between public and private organizations, is to organize the collection and distribution of maps and 3d-models. Consider to focus on critical infrastructure.

For the best response and investigation of a CBRN-incident, a team of specialists in GIS, 3d modelling, visualization and reconstruction has to be organized. This team, that could consist of specialists in the public and private sector as well, should be available for action according to agreements made with the organizations that are responsible for the first response and investigation.

T3.4: Safety risk assessment tool (RAT)
The aim of the development of RAT was to set the use of PPE of the investigators. For instance, at the RN-exercise, the shipping of the exhibits to an external lab was prepared by personnel wearing only Tyvek overalls and Filtering Facepieces as PPE, since a release of the agent could not take place any more due to the double packing at the hot zone. At the Bio-exercise, the CBRN- team entered in a Gas-Tight suite with independent air supply. As it became clear that the agent was indeed Anthrax, the forensic investigators lowered their PPE to the filtered air supplied suites.

The type of PPE worn during the Chemical exercise was determined in the preparation of the exercise using the anticipated outcome of the RAT and the information imbedded in the scenario. The sequence was as follows, see also Fig 3.

1. Reconnaissance by Forensic Incident Commander and Civil Protection Team
Non-permeable Gastight suit and autonomous breathing apparatus (Compressed air)
2. Risk Assessment
3. Sampling traditional exhibits and traces
Non-permeable Tychem suit and gas mask with filter canister (powered ventilation)
4. Risk Assessment
5. Sampling CBRN agents
Semi-permeable ,military suit and gas mask with filter canister (passive ventilation)

T3.5: Visualization of fingerprints
The hyperspectral imaging technology developed during the GIFT project has shown great promise and remains an attractive commercial proposition. It does, however, still require significant development work before a product can be brought to market. The focus will be split over a number of strands: 1) light source, 2) imaging technology, 3) data processing and 4) user properties.
For certain target applications, it may be the case that the eventual product may not require full hyperspectral capability. This would arise for applications that may only call for a basic detection capability at only a few discrete wavelengths. In such a scenario the wavelengths of interest would lie on key spectral features alongside a non-absorbing reference wavelength. This characteristic could then be used to detect the target compounds and to create a differential image using thresholding in the image construction. Such a system could potentially reduce cost or system complexity.
T3.6: Identification and localization of R/N materials by a γ camera

T3.7: Determination of α radiation sources
Alpha particles ionize and excite atmospheric molecules during their passage through air. As a result of this process, some nitrogen molecules are excited to luminous states which relaxate spontaneously within few nanoseconds. The light is emitted by nitrogen molecules in the near ultraviolet (UV) region. The emission spectrum consists of distinct peaks,. The conversion efficiency from kinetic energy into optical radiation is in the order of 10-5 which equals to approximately 100 photons per one 5 MeV alpha particle. Due its low intensity, the UV signal can be overwhelmed by any background light which overlaps with the emission wavelengths. The benefits of the optical approach are the multiplication of signal carriers and significant extension in their range. The attenuation of two UV wavelengths as a function of propagation distance.
In this work, Andor iXon3 897 camera was used for the imaging. It is a scientific-grade EMCCD camera which enables the detection of single photon signals by utilizing on-chip electron multiplication (EM). The multiplication mitigates the effect of sensor read noise by amplifying the pixel signals before analogue to digital conversion. The camera was equipped with a Kokagu lens (UV1228CM – 12mm F/2.8) which can be used down to 300 nm wavelengths.

T3.8: Detection of chemical agents
• A new HRDMA instrument for explosive detection with a resolving power exceeding 40 has been designed and built. The high resolving power achieved allows the separation of samples with very similar mobilities, so the false positive rates could be diminished.
• The HRDMA has been characterised using mobility standards, its resolution and transmission has been determined as a function of sheath flow rate
• Qualitative and quantitative multivariate analysis of the samples have been performed
• Multivariate PCA analysis has allowed the separation of different compounds in clusters
• HRDMA has shown a quantitative behaviour with different organic compounds
• Head space sampling and analysis using UV-DMA has allowed the detection of several explosives, including the lower vapour pressure explosives by only warming up the sample.
• Several explosives, explosive precursors and CWA simulants have been detected using UV-DMA
• Blind samples have been analysed and several explosives have been identified correctly
• Improvements in the sensitivity of the instrument can be implemented
• Integration in the GIFT toolbox and demonstration has been done in the last project months

Crime scene forensics needs to determine on-site the chemical nature of a sample in order to collect it for further laboratory analysis or dismiss it. The instruments currently used for on-site chemical speciation (such as Ion Mobility Spectrometers, Fourier Transform InfraRed spectroscopy, Photoionization Detection, Flame Photometric Detection), do not have sufficient sensitivity or resolving power (or both) and therefore have a very high false alarm rate or are too selective and just detect a certain family of compounds. Therefore there is a need for complimentary technologies that can be used for on-site detection of chemicals that enable preliminary testing to be done at the crime scene.

Tyndall-UCC has focused on the development of miniaturised chemical analysis devices, based on:
a. Micro Total Analysis System (μTAS) devices that will enable flow through miniaturised
capillary electrophoresis (CE) and capillary electro-chromatography (CEC) on-chip with
amperometric detection of chemicals of interest;
b. Impendence sensors that can be used to provide a chemical profile in the development of a
robust and extendible portable sensing platform capable of detection heavy metals

The system has been designed for first responders and forensic investigators for both laboratory andfield detection and identification of dangerous chemicals.

T3.9: Detection of bio agents
Tthe development of a portable and field-applicable PCR based bio-detection unit and initial tests for proof of concept for the functioning of the system. The developed bio-detection unit consists of mainly three parts:
(1) a DNA extraction module,
(2) a PCR module and
(3) a module for visualizing PCR results.

First step in the PCR based detection of biological agents is successful isolation of DNA to be used in the analysis. Successful isolation in terms of PCR includes not necessarily a very high yield but a high quality pure DNA isolate devoid of PCR inhibitors. Certain microorganisms, when faced with harsh environmental conditions, such as low nutrients and water, temperature extremes, etc, form highly durable spores. Those spores are not vegetative structures, which are smaller than the original microorganism and survive in the environment for longer period. Spores are more readily dispersible and can readily turn into vegetative form when encounter favorable conditions.
Detection of biological agents using PCR based techniques is based on amplification and detection of agent-specific sequences, i.e. markers. For this purpose, within the context of this project PCR kits for the detection of B.anthracis and Salmonellae species and a thermal cycler were developed. For field application purposes, both the thermal cycler and the OCR kits were designed to be mobile. For this purpose, ready-to-use PCR kits were prepared. The lyophilized PCR kits require only PCR-grade water and DNA template, i.e. DNA isolate, to be added and run in a thermal cycler.

2. WP4, CBRN Forensic Tools in a Laboratory Environment
The objectives of WP4 are to develop state of the art procedures and methodologies to enable traditional forensic science (DNA, fingerprints and recovery of data from electronic devices) on CBRN contaminated exhibits. And to develop state of the art procedures and methodologies to enable traditional forensic science (DNA, fingerprints and recovery of data from electronic devices) on CBRN contaminated exhibits, focussing on:
• Decontamination
• The forensic investigation of contaminated items under protective conditions (Containment Method)

With respect to the scope of this work package WP4 focused on:
- Traditional forensic investigations within a laboratory environment. The WP did seek to address the efficiency of the decontamination methods currently applied and assessed how these decontaminants affect the recovery of the traditional forensic traces (DNA (blood, saliva), fingerprints (on glass and paper), data from digital data carriers (USB stick, SIM card, mobile phone).
- Traditional forensic investigations through decontamination:

- WP4 did also seek to develop forensic recovery methods that can be applied within a containment method when decontamination is not an option.
- Traditional forensic investigations through containment:

The deliverables of WP4 have all been completed.
• Literature Review regarding decontamination (Report);
• Matrix of experiments – decontamination (Report);
• D4.2 – Literature review regarding FICM (Report);
• Matrix of experiments regarding FICM (Report);
• Experimental and validation work of forensic methods in FICM (Report);
• The developments of decontamination methods, where the Vacuum Decontamination Chamber methods showed the best results in a proof of concept.

3. WP5, CBRN Forensic Investigation
The objective of this Work Package was to develop laboratory methods for profiling CBRN agents released at an incident and to identify signatures for improved attribution to be able to identify the source of chemical, biological and radiological materials used in illegal activities for possibly determine the origin and routes of transit and ultimately contribute to the prosecution of those responsible.
Fig. 5.1 Structure of WP5

5.1.1 Chemical Threat Agents
The aim of the subtask was to develop analytical procedures that provide information about the C-agent itself and secondly to identify the Chemical Attribution Signatures”, CAS, of the agents that may provide sufficient information to relate it to any possible source, perpetrator or vendor.
Three distinct subjects have been identified for development:
• Generic screening analysis of environmental samples
• Generic screening analysis of biomedical samples
• Chemical attribution signatures.

In brief the outline of the activities within subject 1 for the generic analysis of environmental samples that took place were:
• Identification of the gaps in the area of chemical agent analysis in environmental samples:
• based on this, the further development of generic methods for analysis of environmental samples:
• Experiments were performed with 3 model compounds (phorate, fentanyl, acrylonitrile), using different environmental matrices
• Method developed for the generic screening of 250 pesticides

In brief the outline of the activities within subject 2 for the generic analysis of biomedical samples that took place were:
• Human blood samples were exposed in vitro to various concentrations of the 3 model compounds (phorate, fentanyl, acrylonitrile) which were subsequently separated into a soluble part for metabolite/intact agent analysis by means of GC-MS, and into a protein precipitate which was used for protein adduct analysis by means of LC-MS
• For each of the model compounds (i.e. intact agent, metabolite, protein adduct) sensitive analytical methods were developed which eventually can be used for the forensic toolbox (WP 6)

The work on the three topics has been completed by the development of analytical methods which can be used in WP 6. Integration of CBRN Forensic Toolbox:
1. Generic screening analysis of environmental samples
2. Generic screening analysis of biomedical samples
3. Chemical attribution signatures
Including the harmonisation of methods for environmental and biomedical samples.
All is finalized in the D5.5 final deliverable report “Chemical forensic analysis of chemical threat agents”

5.1.2 Toxins
The aim of this task was to develop generic analytical tools for toxins, i.e. toxic biomolecules from living organisms (e.g. plants, fungi and moulds) with the following objectives:
• a general screening method for toxins;
• attribution profiling of toxin samples by performing a study using Amanita mushrooms as model. A complex set of attribution markers will be collected:
• analysis of genetic markers
• toxin and metabolite markers
• stable isotope ratio- and trace metal analyses
• attribution profiling of ama- and phallotoxins in food samples.

The realized work for the analysis of toxins within the GIFT project are:
• to use the established analytical methods on complex sample sets to establish the strength of the general toxin screening and toxin attribution profiling tools;
• Forensic applications:
• Toxin identification in complex sample matrices;
• Source species identification;
• Geographic localization of source plant provenance;
• Toxin sample matching.

5.2.1 DNA preparation – in Bio agents
The aim of this subtask was to develop a tool which comprises a sampling and successive DNA-extraction step, that yields a non-infectious DNA/RNA-extract out of different samples which can be found at a crime scene or other relevant sites. The obtained non-infectious DNA/RNA extract has to meet the criteria which are needed for detection, identification or strain typing of B-agents.
During the project the following tasks have been realized:
- sampling strategy for microbial forensic investigation;
- sampling preparation procedures for molecular analysis of bio agents in a forensic context;
- requirements on sample preparation for different techniques.

A protocol to obtain a non-infectious DNA-extract has been developed and,
- the modus operandi has been tested at the NFI for Lactobacillus casei (non-pathogenic bacteria)
- the protocol to obtain a non-infectious DNA extract consists of three successive steps:
1. DNA-extraction (hand held bead beater)
2. Filtration
3. Addition of antimicrobial peptide
The proof of principle has been tested under laboratory conditions yielding to a non-infectious DNA-extract. During the proof of concept testing of the protocol after each step the survival of L. case and quality/quantity of DNA was tested.

Protocol is described in a concept article to be published:
“Non-infectious DNA-extract from bacteria for microbial forensic investigations”

5.2.2 Identification of genomic signatures for improved attribution of bio agents
The objective of this task was to provide a general statistical method for calculating valid evidence value of source attribution to a pathogen used in an illegitimate context that is accepted and admissible in Court.
To achieve the objectives the following scope has been defined;
• evaluation and development of statistical methods for attribution purposed in the field of microbial forensic data;
• to perform serial transfer experiments;
• to generate a computer program that simulates bacterial growth.

The following activities within this task have been realized and completed in a report:
1. Evaluation and development of statistical methods for attribution purposed in the field of microbial forensic data
• The Evaluation part done
2. To perform serial transfer experiments
• Two runs sequenced and analysed
• Interpretation
• Do we need more runs?
• To generate a computer program that simulates bacterial growth
• Two runs sequenced and analysed
• Interpretation and need for more runs left to do.
3. Development of statistical methods
• Calculating evidence values for scenarios with different statistical methods
• Comparison of methods
4. Simulation program
• Calibration with results from serial transfer experiment
• Validation and testing
5. Serial transfer experiments
• Interpretation

5.3 RN forensic analysis
The objective of this task was to describe the state of the art of Nuclear Forensics analysis in the EU and to compare this with the needs of law enforcement.
The following the objectives have been used to achieve the results for this task:
• a survey of existing laboratory capabilities for nuclear forensics analysis in EU member states;
• a report on nuclear forensics analysis and interpretation of nuclear materials and signatures of radiological materials, related to the information that can be obtained from nuclear forensics analysis and interpretation and how does this match the needs of law enforcement?
The finished activities and perspectives of RN forensic analysis are:
1. Current capabilities available at European laboratories
- Questionnaire sent out to Member States;
- replies received from 19 of 28 Member States;
- currently compiling the report.
2. Nuclear Forensics analysis results and their limitations
- Prepare a draft table of content;
- Intermediate report on nuclear forensics analysis
3. Needs of the law enforcement community
- prepare a draft table of content
- gap analysis
4. Existing mechanisms for mutual support
- report

WP6, Integration of Forensic Toolbox
The objective of this WP was the focus on the interfacing of the GIFT developments in the WP 3, 4 and 5 with the forensic toolbox, therefore:
- liaising with on-going FP7 and also nationally funded project that are or have developed technologies for CBRN detection and to investigate whether these technologies can be applied to the proposed forensic toolbox have been realized.
- a web based forensic toolbox which will link closely with technology proposed in WP 3, 4 and 5 has been developed, demonstrated and validated at the GIFT exercises.
- all aspects of interfacing of the various technologies, sensors and analysis techniques developed in WPs 3, 4 & 5 into system hub for complete total analysis of the information from the CBRN incident has been realized.
To achieve the objectives the following task have been defined and applied:
Task 6.1 Access existing technology developed for CBRN detection;
Task 6.2 Integrated CBRN forensic toolbox system;
Task 6.3 Laboratory requirements for CBRN forensic analysis;
Task 6.4 Web Based Information Hub with Analytics;
Task 6.4.1. Decision Support System;
Task 6.4.2. Information Hub;
Task 6.5 Data transfer and communication with toolbox;
Task 6.6 Software development for data handling.
As WP6 is integrating and interfacing the deliverables of the WPs 3, 4 and 5, alignment and linkage between the WPs is of paramount importance (see figure 6.1)
Figure 6.1

The developed integrated forensic toolbox is illustrated in figure 6.2. This figure shows the initial measurements at the crime scene, the data storage at the toolbox, the knowledge database supplying SOPs for sampling, packaging and transport of CBRN contaminated materials and the risk charts of the identified agent(s). For the evidence send to the laboratory a similar process is described so that the Command and Control Center is provided with the required investigation results to make next steps in the investigation process. The data and results storage does meet all the requirements to maintain the chain of custody.
Figure 6.2

Furthermore a generic communication protocol is developed to accomplish data communication between the GIFT technical deliverables and the toolbox. The data can be transferred through an encrypted USB storage device or through an encrypted internet connection (e.g. satellite, 3G/4G, wifi, wired connections).

To make the measured data available for the investigators, the command and control center a graphic user interface has been developed.

Potential Impact:
Potential impact
On security of citizens: The GIFT project has made common procedures, practices and guidelines, detection systems and laboratory methods for CBRN forensic aspects available. With this the outcome of the project can contribute to a safer and more secure society for the citizens in Europe. As the GIFT work can be applied to provide intelligence services the results of forensic investigation that can be of help of preventing CBRN incidents with a criminal or terrorist intention.
Further the forensic investigation supported by GIFT deliverables can contribute to arrest, prosecute and convict the perpetrators of CBRN attacks. This done on national level, multi- national level or even on request for international organization such as the United Nations, the Organisation for the Prohibition of Chemical Weapons, International Atomic Energy Agency, the Biological and Toxin Weapons Convention and the International Criminal Courts.

Potential users of the expected developments will be public users from the Police, Law Enforcement, Prosecutors, Judges, Forensic Institutes, Defense Organizations, Fire brigade and Paramedics.
Each of these users should be able to adapt and implement the deliverables of GIFT accompanied by education and training of the staff as is developed in the GIFT curriculum appraoach.

The economic impact is difficult to calculate because how to estimate the societal impact and costs to recover society and how to determine the value of citizens feeling safe and secure.
On the level of sme’s some of the deliverables can be commercialised, although it is advised for the sme’s to broaden their scope of the product not only to forensic organisations, but also to defense organisations,, customs, fire brigade etc.

For the forensic community the outcome of the GIFT project is very welcome to gain support within the European Network of Forensic Science Institutes, so that within ENFSI innovations on CBRN forensics are emphasized.