From battlefield to burial: Complex casualty identification using rapid DNA analysis in 21st century warfare

An integratable microfluidic cartridge for forensic swab samples lysis

Decision support for using mobile Rapid DNA analysis at the crime scene

In the 1980s, the advent of DNA analysis revolutionized forensic investigation and has since been the subject of continuous development. As a part of the latest advancements in the field, rapid DNA analysis forecasts a future in which DNA traces can be analyzed at the scene of the crime, in a fully automated manner. Four main platforms developed for rapid DNA analysis include DNAScan, ANDE, RapidHIT 200, and RapidHIT ID. Baring some contrasts such as the number of samples permitted in a single run, they all reflect the distinct characteristics of the current state of rapid DNA analysis technology. Even though time is significantly reduce from days or weeks to a mere 1.5–2 hours, validation studies reveal that the system is not yet sensitive enough for some types of samples commonly found at crime scenes, predominantly those of touched, mixed, or degraded DNA samples. It, however, shows promising results with regards to samples of larger DNA quantities such as blood and saliva. Due to these recognized shortcomings and consequent lack of system validation, no forensic laboratories has thus far adapted this technology as a regularly used tool. Efforts and collaborations by the manufacturers, the FBI, and the Scientific Working Group on DNA Analysis Methods, among others, has long been and are still active in hopes of developing a validated system that will allow the application of rapid DNA analyses in crime scenes, as the situation currently is with higher-quality reference buccal DNA samples.

Three commercially available integrated rapid DNA instruments were tested as a part of a rapid DNA maturity assessment in July of 2018. The assessment was conducted with sets of blinded single-source reference samples provided to participants for testing on the individual rapid platforms within their laboratories. The data were returned to the National Institute of Standards and Technology (NIST) for review and analysis. Both FBI-defined automated review (Rapid DNA Analysis) and manual review (Modified Rapid DNA Analysis) of the datasets were conducted to assess the success of genotyping the 20 Combined DNA Index System (CODIS) core STR loci and full profiles generated by the instruments. Genotype results from the multiple platforms, participating laboratories, and STR typing chemistries were combined into a single analysis. The Rapid DNA Analysis resulted in a success rate of 80% for full profiles (85% for the 20 CODIS core loci) with automated analysis. Modified Rapid DNA Analysis resulted in a success rate of 90% for both the CODIS 20 core loci and full profiles (all attempted loci per chemistry). An analysis of the peak height ratios demonstrated that 95% of all heterozygous alleles were above 59% heterozygote balance. For base-pair sizing precision, the precision was below the standard 0.5bp deviation for both the ANDE 6C System and the RapidHIT 200.

An Oligonucleotide Microarray for High-Throughput Sequencing of the Mitochondrial Genome

Technological innovations enable rapid DNA analysis implementation possibilities. Concordantly, rapid DNA devices are being used in practice. However, the effects of implementing rapid DNA technologies in the crime scene investigation procedure have only been evaluated to a limited extent. In this study a field experiment was set up comparing 47 real crime scene cases following a rapid DNA analysis procedure outside of the laboratory (decentral), with 50 cases following the regular DNA analysis procedure at the forensic laboratory. The impact on duration of the investigative process, and on the quality of the analyzed trace results (97 blood and 38 saliva traces) was measured. The results of the study show that the duration of the investigation process has been significantly reduced in cases where the decentral rapid DNA procedure was deployed, compared to cases where the regular procedure was used. Most of the delay in the regular process lies in the procedural steps during the police investigation, not in the DNA analysis, which highlights the importance of an effective work process and having sufficient capacity available. This study also shows that rapid DNA techniques are less sensitive than regular DNA analysis equipment. The device used in this study was only to a limited extent suitable for the analysis of saliva traces secured at the crime scene and can mainly be used for the analysis of visible blood traces with an expected high DNA quantity of a single donor.

BackgroundShort tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time.ResultsA Low DNA Content BioChipSet (LDC BCS) was developed and manufactured by injection molding. It was designed to function in the fully integrated Accelerated Nuclear DNA Equipment (ANDE) instrument previously designed for analysis of buccal swab and other high DNA content samples (Investigative Genet. 4(1):1–15, 2013). The LDC BCS performs efficient DNA purification followed by microfluidic ultrafiltration of the purified DNA, maximizing the quantity of DNA available for subsequent amplification and electrophoretic separation and detection of amplified fragments. The system demonstrates accuracy, precision, resolution, signal strength, and peak height ratios appropriate for casework analysis.ConclusionsThe LDC rapid DNA analysis system is effective for the generation of STR profiles from a wide range of sample types. The technology broadens the range of sample types that can be processed and minimizes the time between sample collection, sample processing and analysis, and generation of actionable intelligence. The fully integrated Expert System is capable of interpreting a wide range or sample types and input DNA quantities, allowing samples to be processed and interpreted without a technical operator.

Rapid DNA for crime scene use: Enhancements and data needed to consider use on forensic evidence for State and National DNA Databasing – An agreed position statement by ENFSI, SWGDAM and the Rapid DNA Crime Scene Technology Advancement Task Group

A rapid DNA analysis has been developed based on a fluorescence intensity change of a molecular beacon in a PDMS microfluidic channel. Recently, we reported a new analytical method of DNA hybridization involving a PDMS microfluidic sensor using fluorescence energy transfer (FRET). However, there are some limitations in its application to real DNA samples because the target DNA must be labelled with a suitable fluorescent dye. To resolve this problem, we have developed a new DNA microfluidic sensor using a molecular beacon. By monitoring the change in the restored fluorescence intensity along the channel length, it is possible to rapidly detect any hybridization of the molecular beacon to the target DNA. In this case, the target DNA does not need to be labelled. Our experimental results demonstrate that this microfluidic sensor using a molecular beacon is a promising diagnostic tool for rapid DNA hybridization analysis.

A developmental validation was performed to demonstrate reliability, reproducibility, and robustness of the ANDE Rapid DNA Identification System for processing of crime scene and disaster victim identification (DVI) samples. A total of 1705 samples were evaluated, including blood, oral epithelial samples from drinking containers, samples on FTA and untreated paper, semen, bone, and soft tissues. This study was conducted to address the FBI’s Quality Assurance Standards on developmental validation and to accumulate data from a sufficient number of unique donors and sample types to meet NDIS submission requirements for acceptance of the ANDE Expert System for casework use. To date, no Expert System has been approved for such samples, but the results of this study demonstrated that the automated Expert System performs similarly to conventional laboratory data analysis. Furthermore, Rapid DNA analysis demonstrated accuracy, precision, resolution, concordance, and reproducibility that were comparable to conventional processing along with appropriate species specificity, limit of detection, performance in the presence of inhibitors. No lane‐to‐lane or run‐to‐run contamination was observed, and the system correctly identified the presence of mixtures. Taken together, the ANDE instrument, I‐Chip consumable, FlexPlex chemistry (a 27‐locus STR assay compatible with all widely used global loci, including the CODIS core 20 loci), and automated Expert System successfully processed and interpreted more than 1200 unique samples with over 99.99% concordant CODIS alleles. This extensive developmental validation data provides support for broad use of the system by agencies and accredited forensic laboratories in single‐source suspect‐evidence comparisons, local database searches, and DVI.

Rapid identification of human remains following mass casualty events is essential to bring closure to family members and friends of the victims. Unfortunately, disaster victim identification, missing persons identification, and forensic casework analysis are often complicated by sample degradation due to exposure to harsh environmental conditions. Following a mass disaster, forensic laboratories may be overwhelmed by the number of dissociated portions that require identification and reassociation or compromised by the event itself. The interval between the disaster and receipt of victim samples at a laboratory is critical in that sample quality deteriorates as the postmortem interval increases. When bodies decompose due to delay in collection, transport, and sample processing, DNA becomes progressively fragmented, adversely impacting identification. We have previously developed a fully automated, field-forward Rapid DNA identification system that produces STR profiles (also referred to as DNA IDs or DNA fingerprints) from buccal and crime scene samples. The system performs all sample processing and data interpretation in less than 2 h. Here, we present results on Rapid DNA identification performed on several tissue types (including buccal, muscle, liver, brain, tooth, and bone) from exposed human bodies placed above ground or stored in a morgue/cooler, two scenarios commonly encountered following mass disasters. We demonstrate that for exposed remains, buccal swabs are the sample of choice for up to 11 days exposure and bone and tooth samples generated excellent DNA IDs for the 1-year duration of the study. For refrigerated remains, all sample types generated excellent DNA IDs for the 3-month testing period.

Rapid DNA analysis is an ultrafast and fully automated DNA-typing system, which can produce interpretable genetic profiles from biological samples within 90 minutes. This “swab in—profile out” method comprises DNA extraction, amplification by PCR multiplex, separation and detection of DNA fragments by capillary electrophoresis. The aim of study was the validation of the Accelerated Nuclear DNA Equipment (ANDE) 6C system as a typing method for reference samples according to the ISO/IEC 17025 standard. Here, we report the evaluation of the validity and reproducibility of results by the comparison of the genetic profiles generated by the ANDE 6C System with those generated by standard technologies. A quantity of 104 buccal swabs were analyzed both through the ANDE 6C technology and the traditional method (DNA extraction and quantification, amplification and separation by capillary electrophoresis). Positive typing was observed in 97% of cases for ANDE 6C technology with only three buccal swabs failing to reveal interpretable signals. Concordance was determined by comparing the allele calls generated by ANDE 6C and conventional technology. Comparison of 2800 genotypes revealed a concordance rate of 99.96%. These results met the ISO/IEC 17025 requirements, enabling us to receive the accreditation for this method. Finally, rapid technology has certainly reached a level of reliability which has made its use in laboratories of forensic genetics a reality.

We report the design and performance validation of microfluidic separation technologies for human identification using a disposable plastic device suitable for integration into an automated rapid DNA analysis system. A fabrication process for a 15-cm long hot-embossed plastic microfluidic devices with a smooth semielliptical cross section out of cyclic olefin copolymer is presented. We propose a mixed polymer solution of 95% w/v hydroxyethylcellulose and 5% w/v polyvinylpyrrolidone for a final polymer concentration of 2.5 or 3.0% to be used as coating and sieving matrix for DNA separation. This formulation allows preparing the microchip without pretreatment in a single-loading step and provides high-resolution separation (≈1.2 bp for fragments <200 bp), which is superior to existing commercial matrices under the same conditions. The hot-embossed device performance is characterized and compared to injection-molded devices made out of cyclic olefin copolymer based on their respective injector geometry, channel shape, and surface charges. Each device design is assessed by fluorescence videomicroscopy to evaluate the formation of injection plugs, then by comparing electropherograms for the separation of a DNA size standard relevant to human identification.

This chapter sees the partially realised nature of these technologies as an opportunity rather than a problem. It utilises this opportunity to explore interpretations of emerging forensic DNA technologies to illuminate how they render orderings of various domains fluid and malleable. Such domains include the natural and social sciences, ethics, law, commerce and society at large. The chapter draws on the conceptual framing of interpretive flexibility as developed via social construction of technology studies, which have highlighted the different perceptions of technology on the part of different stakeholders embedded in sociotechnical networks. Differing standpoints and interpretations of technology may lead to contested plans and imaginaries. Framing technolegal anticipation (namely, anticipations of law-science-technology-society relations) in terms of embedded interpretive flexibility enables the chapter to illuminate differing normative interpretations of technology and sociotechnical engagement. Here, discursive responses to three notable examples of emerging forensic DNA methods are explored: rapid DNA analysis, forensic DNA phenotyping (FDP) and forensic genealogical testing. These three examples are presented in order to examine how their perceived status embodies differing assumptions concerning how science and wider society should engage, which, in turn, order relations between domains in different ways.

Chapter 16 - Emerging technologies for DNA analysis of challenged samples