‌Improved Methods for Transport and Preservation of DNA Samples for Victim Identification in a Military Environment‌

Abstract

Identification of military casualties resulting from a disaster or mass fatality event while deployed to an area of operations is facilitated by the Mortuary Affairs Forensic Response Team (Mortuary Affairs) using methodologies such as DNA, fingerprints and dental examination. These scientific methods have been successfully used in many civilian disasters to identify victims; however, military environments present extra logistical challenges that need consideration. Preservation of bone and soft tissue samples during transport is necessary to prevent degradation prior to DNA profiling. Currently Acu- Temp AX56L mobile battery-operated refrigerator/freezers are deployed as part of the Mortuary Affairs response, although the units are not practical for a rapid field response and transport from remote sites, including combat zones.

An alternative DNA preservation method is required by the ADF to guarantee efficient warfare logistics support for victim identification suitable for any scenario involving military deaths.

Image courtesy Department of Defence

This article examines the constraints of the current DNA preservation method in a military context and suggests alternatives that may improve logistics support for response to military casualties. Three chemical preservatives were used on fresh and partially decomposed bone and muscle samples stored at 21°C, 45°C, 55°C and 65°C for one week. The samples were DNA profiled and found to be the same quality as those obtained from refrigerated samples. The preservatives are cheap and lightweight and meet user requirements for the Forensic Identification Equipment Immediate Response (fly-away) Kit and the Follow-on Response Kit.

Disaster Victim Identification

Disaster victim identification (DVI) is the structured response to a single or mass casualty event caused by an accident, a natural disaster or a deliberate act to take lives. In a military context the events requiring a DVI response may involve deaths from land, aerial or naval warfare; deaths in training accidents; response to humanitarian disasters; or situations where the government requires support for a civilian disaster. Government agencies worldwide have adopted the International Criminal Police Organisation (INTERPOL) Disaster Victim Identification Guide,¹ which outline processes incorporating scientific and investigative principles that are collectively applied to identify victims, who are then returned to loved ones. The nature of some disasters prevents visual identification, so proof of identity is achieved using the ‘primary identifiers’—fingerprints, dental and DNA. A match between an ante-mortem (AM) record and a post-mortem (PM) record by any one of these methods may be accepted as sufficient evidence for identification. Property (such as jewellery, wallets and clothing), and physical evidence (such as scars, tattoos and medical implants) are ‘secondary identifiers’, which cannot be used solely to identify a victim. They can, however, target the use of primary identifiers and support identification.

A DVI operation consists of the recovery phase, the AM and PM phases, and the reconciliation and repatriation phases. The recovery phase involves the recording and collection of evidence and bodies from the disaster site once it is practical and safe. The recovered remains are transported to a mortuary facility, where any associated property is recorded and preserved, physical evidence and fingerprints are recovered, dental X-rays and examinations are performed, and DNA samples are collected (the PM phase). The AM phase comprises recording information about a missing person and collating the evidence that could be compared with PM evidence recovered from the remains. Investigators and scientific experts conduct the reconciliation phase, where AM and PM evidence is systematically compared. An identification report is prepared when a match is found that meets a specified threshold. This report is then reviewed independently against all other information pertaining to the case by an investigator in the reconciliation team. A reconciliation comparison report is then provided to an identification board—a group of experts who have the authority to make decisions on identification. The formal release and transfer of the body from the mortuary is known as the repatriation phase. INTERPOL DVI forms² (electronic or hard copy) are used throughout each phase to record information in a standardised approach that facilitates comparison of information.

While the DVI process outlined above is generally the same, each disaster may have its own characteristics and therefore requires a tailored response. The Australian Defence Force (ADF) Joint Services Police Group has trained personnel who generally will be called upon to conduct the recovery phase in a military-assisted or combat DVI operation; however, particularly in a combat environment, body recovery may be conducted by other personnel. Fingerprints and dental methods are reliable, and often faster than DNA profiling, at achieving identification. When fingerprint and dental identification is not possible due to the condition of the remains, DNA is heavily relied upon as the only remaining primary identifier. The 2001 World Trade Centre (WTC) DVI operation posed issues due to the high impact forces of the planes, collapse of both buildings and the resulting inferno, all of which resulted in highly fragmented and co-mingled remains; and it was considered an ‘open disaster’, meaning authorities did not know exactly how many victims required identification. Open disasters often require more extensive recovery and PM phases to ensure all victims are accounted for. Considering the WTC attack was an open disaster, the nature of the attack and the high degree of body fragmentation, authorities directed the DVI operation to DNA test all remains recovered from the site, including pieces of tissue only centimetres in size. A total of 2,749 people were killed in the attack, but they were represented by over 20,000 PM samples. The recovery phase continued for months after the event, hindered by the slow removal of rubble, which was then sifted through by hand (twice) to find remains. The DNA testing laboratory was close to the recovery site. This reduced the logistics complexity for sample preservation and transport. Nonetheless, it highlights the potential volume of DNA samples arising from high-impact open disasters.

The 2002 Bali bombings was also an open disaster and involved a high degree of body fragmentation and some co-mingling. The combined attack resulted in 202 victims, representing 23 nationalities. Authorities decided that all remains should be tested for DNA; however, a suitable DNA laboratory was not available in Indonesia to conduct the analysis. The DNA samples were collected at the mortuary site in Bali and transported in polystyrene boxes packed with dry ice. A total of 1,046 PM DNA samples needed transportation from Bali to the Australian Federal Police laboratory in Canberra after the then Prime Minister, the Hon. John Howard MP, offered to sponsor DNA testing and all associated costs, regardless of victim nationality. The samples were sent to Australia in batches using commercial carriers and ADF assets which sufficiently preserved the DNA. Some extra consideration was needed to transport the dry ice by air given its classification as dangerous goods—specifically, the samples must be packaged to allow release of carbon dioxide gas, and some carriers have a weight limit for carry-on and checked packages containing dry ice (Qantas currently allows only 2.5 kilograms per passenger).³ This was one of the first examples where ADF assets were needed to transport DNA samples from an overseas DVI operation. While dry ice was readily available in this scenario, it is not expected in remote sites, and its transportation by air introduces logistical complexities.

The 2004 Boxing Day tsunami killed over 250,000 people in 13 countries. The DVI response in Phuket, Thailand, comprised scientists, police and support staff from over 40 nations. A total of 3,679 victims, representing 42 nationalities, required identification. Victims’ bodies were not fragmented, limiting the number of DNA samples required for each victim to only one or two. The samples were primarily analysed in China, Sweden and Bosnia and Herzegovina, requiring multiple flights and ground transport transfers from Thailand. Germany, Australia and Great Britain also provided DNA testing early in the operation. The samples were stored in specimen jars sealed inside large polystyrene boxes cooled by ice bricks. The ice bricks needed replacement between certain transfers. This involved opening each sealed box, repacking it with frozen ice bricks and resealing it. Although this was an effective method, it increased the complexity of logistics and evidence continuity, which impacted on time and cost efficiencies.

Royal Australian Navy sailor, Leading Seaman Imagery Specialist Jake Badior, documenting the damage in Palu following an earthquake and tsunami as part of Operation Indonesia Assist 2018. Image courtesy Department of Defence

While these are examples of civilian disasters, the ADF may need to respond to similar events that present in a military context, such as aviation and nautical disasters, deaths from improvised explosive devices and terrorist attacks. Deaths from hostile action may involve a high impact element leading to body fragmentation and possible co-mingling of remains. An extra challenge to the identification operation may present if remains of Australian service personnel are co-mingled with remains of victims from another nation, who may be civilians or part of allied or enemy forces. The logistical complexities of DNA sample transport and preservation may be compounded by the operating environments in modern combat zones and the potentially unique and more challenging disaster scenarios encountered by the ADF when compared with civilian DVI operations. The reduced access to permanent facilities, electricity and remote operating environments may hinder DNA sample preservation by mechanical refrigeration or ice. An alternative DNA preservation method is required by the ADF to guarantee efficient warfare logistics support for victim identification suitable for any scenario involving military deaths.

DNA Profiling and Sample Preservation

DNA samples collected from a body during the PM phase of a disaster (civilian or military) preferably consist of either a section of bone (typically no more than five centimetres in length), a tooth, blood or a section of muscle (approximately five millimetres squared). The DNA is extracted from this sample in a forensic biology laboratory, and sections of the genome that are highly variable between individuals are analysed. The resulting DNA profile is compared with the DNA profile from the AM sample—typically a personal effect from the missing person (for example, a toothbrush) or DNA from close biological relatives (for example, parents and siblings). When all highly variable sections of the DNA are successfully profiled (for Australia at least 21 DNA segments), the chance of obtaining an identification when compared with an appropriate AM sample is almost guaranteed.

If the DNA segments of interest are damaged by decomposition, incineration or other adverse environmental factors (known as degradation), they cannot be compared with the AM DNA profile. The chance of obtaining an identification decreases as the number of damaged DNA segments increases. DNA degradation may also occur if the samples are not preserved after removal from the body during the PM phase and during transport to the testing laboratory (typically by mechanical refrigeration or by ice to ~4°C). Without refrigeration, the DNA sample could degrade within hours in certain environments; therefore, continuous sample preservation from collection and during transport is essential. If the DNA samples degrade during transport, and dental and fingerprint evidence is unavailable, the body may have to be re-examined so another sample can be collected. This could delay victim identification for a number of weeks or prevent identification when a second sample is not available (in cases involving a high degree of fragmentation).

Logistical Factors and Operational Considerations

The ADF responds to military personnel involved in a disaster event overseas while deployed to an area of operations. The ADF may also be called upon by the government to supplement a DVI response provided by other Australian agencies in a non-operational area (for example, the 2002 Bali bombings). A designated Forensic Response Team (FRT), part of an ADF Mortuary Affairs cell, responds by travelling from Australia to either an existing mortuary facility near the incident site or to construct a temporary mortuary. The FRT typically consists of a mortuary manager, a forensic pathologist, a forensic dentist, a fingerprint expert, a forensic biologist and a photographer, all of whom will perform the PM phase of the DVI operation. The number and type of personnel required will be assessed on a case-by- case basis, depending largely on the size of the incident.

Forensic Identification Equipment (FIE) is transferred from Australia to the mortuary site in an Immediate Response (fly-away) Kit, with a Follow- on Response Kit transported to sustain the response as required. The equipment includes a forensic identification suite (comprising autopsy instruments, a lighting system and DNA sample refrigeration), digital dental X-ray systems, X-ray equipment to ensure bodies are clear of unexploded ordnance, body bags, storage, computer equipment, photographic equipment and a temporary body storage unit. Ideally the mortuary and equipment should be set up within one to two hours of arrival by the FRT.

The FIE needs to be lightweight (capable of a two-person lift to a maximum of 32 kilograms), simple to use and maintain in harsh environments, available at short notice, reliable, capable of long-term storage and suitable for maintenance or replacement in isolated conditions. It must also be compact, robust and stable, and it must enable users to perform forensic identification procedures, including body preservation, examination, documentation and analysis of AM and PM evidence. The equipment may need to be transferred to a ship and must therefore be suitable for carrying through narrow corridors. It may also require transportation via helicopter, jackstay (flying fox), in a truck over rough roads and cross-country. It should be compact and light for air transportation by rotary and fixed-wing aircraft or to be underslung by helicopter and by rail as part of a freight load, and it should be suitable as checked luggage on commercial aircraft.

The DNA samples collected during the PM phase by the FRT need to be transported from the mortuary to a nominated laboratory in Australia. Sample preservation throughout this period is essential. The total time from mortuary to laboratory could range from hours to days depending on the location of the incident, transport available and other operational constraints, including those experienced in or near combat zones. Blood samples are preserved using Flinders Technology Associates (FTA) paper, which meets FIE requirements and which will continue to be used. The FTA paper fits inside a small envelope and preserves blood spotted onto chemically treated paper without the need for refrigeration for up to 14 years; however, it can only be used if blood is available from remains. Currently Acu-Temp^® Hemacool AX56L mobile battery-operated refrigerator/freezers are used by the ADF to preserve DNA samples (all samples except blood); however, they do not meet the FIE user requirements. Typically, one unit is required for the Immediate Response (fly-away) Kit and two units are required for the Follow-on Response Kit. The unit weighs 65.9 kilograms empty and needs two people to carry it. Its external dimensions are 99.6 centimetres by 58.9 centimetres by 57.9 centimetres (length, width and height), and the payload dimensions are 45.7 centimetres by 35.6 centimetres by 33.0 centimetres squared (56 litres).⁴ Battery life at a storage temperature of 2°C to 8°C is 14 hours at 43°C ambient temperature and 48 hours at 25°C ambient temperature, with auxiliary power including adaptors suitable for air carriers (12-30VDC input power and 100-250VAC power). The cost of each unit is approximately $10,000, and they require regular maintenance.

The size and weight of the units, the small payload and reliance on limited battery life and electricity are the major constraints to using the Acu-Temp® Hemacool AX56L refrigerator/freezers for DNA sample preservation as part of an ADF disaster response strategy. The small payload limits the number of samples that can be transported (one unit may carry as few as 50 bone samples). In a high-impact disaster where fragmentation could be expected, one unit may carry only a fraction of the samples required. Additional units would therefore be required, further increasing the weight and size and exceeding the constraints of the FIE, thus increasing logistics complexity. If the number of casualties or degree of fragmentation is unknown and only one unit is deployed, the lack of preservation for samples exceeding the payload of one refrigerator/freezer unit would prevent shipment of additional DNA samples to Australia and significantly extend the DVI operation out of country, potentially delaying victim identification. A civilian disaster one of the authors worked on involved five victims on a light plane that crashed into a field, resulting in 140 samples that required DNA testing. If this same scenario presented in a military context, the number of victims may initially indicate that only one refrigerator/freezer unit is required; however, given the degree of fragmentation, it is likely that two or three units would be required to transport the DNA samples to Australia using the current ADF strategy. In this scenario, three full units would exceed 200 kilograms. Increasing the number of refrigerator/freezer units also limits the types of transportation options available (land, sea and air) that could provide the necessary auxiliary power when battery life is exceeded, creating further logistical issues. Often the degree of fragmentation may be unknown at the time of equipment deployment, so a scalable sample preservation method is needed.

The weight and size of the unit/s makes some transportation methods unviable, as they exceed the desired footprint of the FIE and consume most of the FRT’s manpower in carrying requirements. The limited battery life may cause logistical challenges when auxiliary power is unavailable or when transport between the mortuary and an electricity source exceeds battery capabilities. There is also the risk of unit malfunction, which may then cause sample degradation if another refrigeration source is unavailable. In areas of operation with extreme heat conditions, the battery life will be severely reduced. In the Middle East, for example, temperatures may reach 53°C and, after fitting vehicles with armour, temperatures inside those vehicles would be expected to exceed outside temperatures. The tarmac or concrete used on airfield aprons reflects heat and further multiplies the temperatures of vehicles on the ground; therefore, sample preservation is needed in environments that could reach or even exceed 60°C.

Proposed Alternative DNA Preservation Strategy

Research has demonstrated that various chemicals can preserve DNA samples without refrigeration. These include ethanol, GenoFix^TM, TypiFix^TM, dimethyl sulfoxide (DMSO), DNA Genotek Tissue Stabilising Kit, Biomatrica^®, DNAgard, Lysis Storage and Transport buffer (LST), and sodium chloride (NaCl). A study by Sorensen et al. (2016)⁵ reported preservation of skin and muscle at 35°C for up to three months. A search of current literature shows that the hottest temperature at which DNA samples were successfully preserved using chemical media was 37°C for 38 weeks.⁶ The most important consideration when selecting a chemical for DNA preservation is that it consistently enables a DNA profile to be obtained from the sample. Other considerations include cost, weight, availability, shelf life, ease of preparation, health hazards, disposal requirements, and stability during transport. Using these criteria, DMSO, ethanol and NaCl were chosen as possible alternatives to refrigeration. Allen-Hall (2011)⁷ found that each chemical preserved fresh human muscle at 35°C for one month. Szibor et al. (2008)⁸ found that NaCl preserved muscle and skin at 37°C for 38 weeks, and Caputo et al. (2011)⁹ found that NaCl preserved human muscle at room temperature (temperature not specified) for one year.

The cost per sample of each chemical is 6.5 cents for NaCl, 0.5 cents for ethanol and 0.9 cents for DMSO (in a two-millimetre collection tube). The weight of a standard bag of NaCl is five kilograms, and a 2.5-litre bottle of ethanol or of DMSO weighs less than three kilograms. The shelf life of NaCl and ethanol (undiluted) is three to five years, and for DMSO it is two years. There are negligible health risks when using each chemical, and they are all readily available and easy to prepare, with minimal equipment required. All chemicals are permissible to transport by air, with NaCl and DMSO not regulated as dangerous goods. Ethanol has dangerous goods restrictions for air travel. Commercial carriers require ethanol storage in leakproof containers or heat-sealed plastic bags packed in 30-millilitre quantities, with a limit of one litre for commercial flights (checked baggage or carry-on luggage).¹⁰ The restricted ethanol volume of one litre is sufficient to preserve hundreds of DNA samples and therefore remains a viable preservative for the FIE.

The chemicals seem promising as a replacement for refrigeration; however, their use to date has been for civilian medical and research applications, and their specifications may not meet military requirements. There is no research to demonstrate whether the chemicals would be suitable for the potentially extreme temperature conditions encountered by the ADF or whether they can preserve tissue with early signs of degradation (which may be expected in a disaster scenario). Therefore, these alternative preservation methods cannot be recommended to the ADF to replace the refrigerator/freezers unless research is conducted that specifically tests the chemicals in military environments.

Testing Preservatives at Extreme Temperatures for Military Applications

The preservation capabilities of DMSO, ethanol (70 per cent) and NaCl were compared against refrigeration for muscle and bone samples. The three chemicals must be able to preserve the samples as well as refrigeration for them to be considered a suitable replacement. Preservation success was measured by DNA profiling each sample using 21 DNA segments and counting how many segments could be reported. Temperatures well above those previously reported for these chemicals were trialled to mimic potential ADF conditions. The temperatures used were 45°C, 55°C and 65°C, with 21°C included as a control to verify the chemicals were performing at standard conditions. It was hoped that the samples stored at 45°C would produce complete DNA profiles (21 out of 21 segments); however, muscle samples stored at 55°C and 65°C were not expected to preserve the DNA anywhere near as well as refrigeration.

Both fresh and decomposed bone and muscle were tested to determine if early stages of tissue decomposition affected the preservation process.,A total of 78 muscle and 78 bone samples were collected from a single donor;¹¹ half of the samples were stored at 30°C for 48 hours to induce early stages of decomposition. Each sample was then preserved in one of the three chemicals for one week at one of the four temperatures nominated above in replicates of three. It was believed that one week would exceed the time required to transport samples from an overseas mortuary to Australia.

The alternative DNA sample preservation strategy using a readily available chemical is suitable for use in a wider range of environmental conditions, including extreme temperatures and remote combat zones scenarios. Image courtesy Department of Defence

Fresh and decomposed muscle and bone were also refrigerated (–4°C) and expected to provide complete DNA profiles.

The refrigerated samples produced complete DNA profiles as expected. The three chemicals preserved DNA samples (fresh and decomposed muscle and bone) as well as refrigeration at all temperatures tested. All samples produced DNA profiles without any signs of degradation, suggesting that they would be suitable for comparison against AM profiles. These results were largely unexpected, particularly for the muscle samples stored at 55°C and 65°C for one week. Such extreme conditions were expected to partially degrade the DNA at those temperatures. The surprising robustness of the preservatives at high temperatures (nearly double the temperature of previous experiments) looks encouraging for use across current and predicted areas of operation, but further research will be needed to validate the method. It was also encouraging that early stages of tissue decomposition did not affect the preservation process with any of the chemicals, further increasing the potential scope of use in an operational context.

Conclusion

This research demonstrates that alternative DNA sample preservation strategies using readily available chemicals may be suitable replacements for refrigerator/freezer units, which are not appropriate for inclusion in the FIE for a DVI response. It is recommended that a further validation trial be conducted prior to operational implementation, with more samples subjected to further temperature variations and additional tissue decomposition methods. The three chemicals tested all meet the FIE user requirements, overcome previously discussed constraints, and offer significant logistical efficiencies. Table 1 below illustrates the cost and weight savings of the chemicals compared with the refrigerator/freezers and highlights the reduced logistics complexity of DNA sample transport when auxiliary power is not required and battery life is not a limiting factor. The chemicals are lightweight and can be readily deployed in the Immediate Response (fly-away) Kit in quantities sufficient for increased scalability if the degree of body fragmentation or number of victims are greater than expected. The chemicals are also readily available and do not require maintenance.

Table 1. Efficiencies of an Alternative DNA Sample Preservation Strategy for a DVI Response

*Two units are required in the FIE kits. b Battery life at nominated ambient temperature.

This research has potential to remove an impediment to the ADF response to a disaster event and improve logistics support for identification of military casualties. The alternative DNA sample preservation strategy using a readily available chemical is suitable for use in a wider range of environmental conditions, including extreme temperatures and remote combat zones, and does not rely on battery or auxiliary power. Chemical preservatives could increase the range of transportation options compared with refrigerator/ freezers and may also reduce the length of a DVI operation. This article is the result of research carried out with the assistance of an Army Research Scheme grant in 2016–17.

Endnotes

1. INTERPOL, 2017, Disaster Victim Identification Guide, Mar, at: https://www.interpol.int/ INTERPOL-expertise/Forensics/DVI-Pages/DVI-guide
2. INTERPOL, 2017, ‘DVI Forms’, Mar, at: https://www.interpol.int/INTERPOL-expertise/ Forensics/DVI-Pages/Forms
3. Qantas, 2017, ‘Dangerous Goods’, Apr, at: https://www.qantas.com/travel/airlines/ dangerous-goods/global/en#carbon-dioxide-solid-dry-ice
4. CSafe Global, 2017, ‘CSafe Global AX56L Product Sheet’, Mar, at: http://csafeglobal.com/ wp-content/uploads/2016/06/CSafeGlobal-AX56L-Product-Sheet-6_03_16-web.pdf
5. A Sorensen, E Rahman, C Canela, D Gangitano and S Hughes-Stamm, 2016, ‘Preservation and Rapid Purification of DNA from Decomposing Human Tissue Samples’, Forensic Science International: Genetics Vol 25 pp 182-90
6. R Szibor, W Huckenbeck, W Thiel, D Krause and R Lessig, ‘Thoughts for the Organisation of an Early Phase Response to Preserve Victim Identification Information After Mass Disasters’, in M Prinz, A Carracedo, WR Mayr, N Morling, TJ Parsons, A Sajantila, R Scheithauer, H Schmitter and PM Schneider, 2007,‘DNA Commission of the International Society for Forensic Genetics (ISFG): Recommendations Regarding the Role of Forensic Genetics for Disaster Victim Identification (DVI)’, Forensic Science International: Genetics Vol 177, pp e39–e42
7. A Allen-Hall, 2011, ‘Preservation of Human Muscle in DVI Conditions Commonly Associated with Mass Disasters’, Honours: University of Canberra, 2011
8. Szibor et al., 2008.
9. M Caputo, L Bosio and D Corach, 2011, ‘Long-term Room Temperature Preservation of Corpse Soft Tissue: An Approach for Tissue Sample Storage’, Investigative Genetics Vol 2 at: https://doi.org/10.1186/2041-2223-2-17
10. Virgin Australia, 2017, ‘Dangerous Goods’, Apr, at: https://www.virginaustralia.com/au/en/ plan/baggage/dangerous-goods/
11. Acquired through the University of Technology Sydney Body Donation Program (UTS HREC, ETH15-0029).