The collection, preservation, and processing of DNA samples from decomposing human remains for more direct disaster victim identification (DVI)

Forensic DNA analysis plays a vital role in forensic casework and mass fatality incidents to identify victims. In these situations, human remains are often subjected to extreme heat, humidity, possible mutilation or fragmentation, microbial activity, decomposition and putrefaction, which can all complicate disaster victim identification (DVI). In many cases, the disaster event results in damage to the local infrastructure causing a loss of electricity and lack of facilities with refrigeration to house large numbers of human remains. Without refrigeration, the DNA in tissues become more degraded and damaged making traditional short tandem repeat (STR) typing more difficult. Therefore, immediate and simple in-field collection and stabilization of DNA samples from decomposing human remains without the need for refrigeration would be of great benefit to the forensic community. In addition, quick victim identification is also a goal for DVI operational teams as they attempt to bring closure to the victim’s families, and assist government agencies that must account for the deceased. As a result, the demand for a rapid turnaround time is often stressed; however, due to the overwhelmingly large number of samples requiring processing, this may not be feasible. Significant rate-limiting steps in the STR typing workflow are DNA extraction (approx. 3-4 hours), DNA quantification (1-2 hours), and standard PCR amplification (approx. 2-3 hours). Digestion of dense connective tissues such as skin and muscle prior to DNA purification may add another 8-12 hours, or hard tissues such as bone or teeth another 24-48 hours to the overall time required for generating an STR profile. If some of these procedures could be reduced (or avoided) during a mass fatality incident involving thousands of victims, sample throughput could be substantially increased. This doctoral research focuses on testing various in-field DNA collection and room temperature preservation methods for decomposing human remains as mock DVI samples. We compared rapid DNA purification protocols or direct amplification approaches that will eliminate unnecessary steps in the DNA analysis workflow, increasing the throughput and reducing the costs of analysis. Overall, results indicate that sufficient DNA can be collected and preserved at ambient temperature using some of these methods, provided that DNA is not already severely degraded before collection. In-field sample collection from human remains using biopsy punches or simply making an incision in the skin and swabbing the underlying muscle with cotton or foam swabs proved to be the most successful and easiest methods of DNA collection. Biopsy punches should be immediately stored in a modified TENT preservative, and swabs allowed to dry for transport to the laboratory for refrigeration, or stored until DNA analysis is possible. TENT and swab samples were processed with both traditional DNA analysis workflows and a direct PCR approach. In all cases, complete profiles were obtained from fresh tissues using all methods tested in this study. However, severely decomposed tissues were more challenging, with most samples yielding partial (or no) STR profiles. We proposed a method for triaging swab samples based on the quantification results of samples prepared for direct PCR in order to increase the first-pass success rate. Results indicate that foam swabs used to collect from muscle tissue may generate the most complete STR profiles for the majority of decomposed tissues, with cotton swabs yielding similar results. In addition, aliquots of TENT containing DNA leached from tissues were successfully diluted and directly added to the PCR reaction, thereby skipping DNA extraction and quantification all together. This protocol is the quickest of all methods tested, generating STR profiles in a fraction of the time it takes for traditional DNA processing. If this first-pass approach fails due to insufficient amounts of DNA, then the tissue itself stored in the TENT buffer can be quickly extracted in under 20 minutes with the PDQeX DNA extraction system. We found that this method generated the most complete STR profiles from severely decomposed tissues.
Overall, we have demonstrated that tissues preserved in a modified TENT buffer or collected and stored using cotton and foam swabs show potential as alternate methods for the immediate in-field collection and preservation of DNA at room temperature for human identification purposes. However, these methods warrant further investigation to optimize protocols to achieve more efficient DNA preservation and higher STR success rates from severely decomposed human tissues.