Investigations into Enhancing Yersinia pestis Cells Viability following Environmental Sampling for Forensic Analysis

Following an intentional or accidental bio-warfare agent (BWA) release, environmental sample analysis is absolutely critical to determine the extent of contamination. When dealing with nonspore forming BWA (e.g., Yersinia pestis), retention of cell viability is central to such analyses. Even though significant advances have been achieved in DNA sequencing technologies, a positive identification of BWAs in environmental samples must be made through the ability of cells to form colony-forming units upon culturing. Inability to revive the cells between collection and analysis renders such studies inconclusive. Commercial kits designed to preserve the viability of pathogens contained within clinical samples are available, but many of them have not been examined for their ability to preserve samples containing suspected BWAs. The study was initiated to examine the applicability of commercial solutions aiding in retention of Y. pestis viability in samples stored under nonpermissive temperatures, that is, 40 and 37°C. While none of the tested solutions sustained cell viability at 40°C, the results show five out of 17 tested preservatives were capable of supporting viability of Y. pestis at 37°C.     

The Optimal Combination of Cartilage Source and Apparatus for Long‐Term In Vitro Chondrocyte Viability Analysis

Abstract: Most studies of long‐term chondrocytes survival were for tissue banks. They showed a gradual reduction in the viable chondrocytes percentage as a function of time and ambient temperature, but the samples were harvested under optimal conditions. The aim of our study was to determine the most reliable combination of cartilage source and assay for the in vitro postmortem chondrocyte viability analysis in the conditions that imitate a dead body. Osteochondral cylinders were procured from femoral condyles and talar trochleas of three male donors and stored in the cell culture media at 4 ± 2°C and 23 ± 2°C. The samples were analyzed by a cell viability analyzer and a confocal laser scanning microscope (CLSM) initially 24–36 h after death and then in 4‐week intervals. The results reconfirmed the significant influence of time (p = 0.0002), but not of the temperature (p = 0.237). The largest reproducibility was presented for the knee joint and the CLSM.     

Species Identification of Cannabis sativa Using Real‐Time Quantitative PCR (qPCR),

Most narcotics‐related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3′ exon‐trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real‐time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa.     

Detection of Anthrax and Other Pathogens Using a Unique Liquid Array Technology

A bead‐based liquid hybridization assay, Luminex^® 100?, was used to identify four pathogenic bacteria, Bacillus anthracis, Clostridium botulinum, Francisella tularensis subsp. tularensis, and Yersinia pestis, and several close relatives. Hybridization between PCR‐amplified target sequences and probe sequences (located within the 23S ribosomal RNA gene rrl and the genes related to the toxicity of each bacterium) was detected in single‐probe or multiple‐probe assays, depending on the organism. The lower limits of detection (LLDs) for the probes ranged from 0.1 to 10 ng. Sensitivity was improved using lambda exonuclease to digest the noncomplementary target strand. All contributors in 33 binary, ternary, and quaternary mixtures in which all components were present in a 1:1 ratio were identified with an 80% success rate. Twenty‐eight binary mixtures in which the two components were combined in various ratios were further studied. All target sequences were detected, even when the minor component was overshadowed by a tenfold excess of the major component.     

Viability of Human Articular Chondrocytes Harvested Postmortem: Changes with Time and Temperature of In Vitro Culture Conditions

Different studies of long‐term chondrocytes viability have shown a gradual reduction as a function of time and ambient temperature. The aim of our in vitro study was to establish chondrocyte postmortem viability curves for 4°C, 11°C, 23°C, 35°C during 63 days after the donors' death. Osteochondral cylinders were procured from the knees of 16 male donors (20–47 years), stored in preservation media that was not changed, and analyzed in 3‐day intervals using a confocal laser scanning microscope. A significant influence of time on viability was found from Day 9 (p = 0.0029) and onwards (p < 0.0001). The lowest overall chondrocyte viability was at 35°C, followed by 4°C (p < 0.0001). The conditions used in this in vitro analysis suggest that similar viabilities may occur while in situ in the decedent. Further studies of chondrocyte viability from individuals with known postmortem intervals may show premise to help evaluate time since death in the late postmortem interval.     

Elemental Analysis of Variably Contaminated Cremains Using X‐ray Fluorescence Spectrometry

Analyzing and identifying skeletal remains becomes increasingly difficult when remains have been cremated, especially in cases where the cremated material may have been intentionally contaminated with nonskeletal material. This study examined the potential of X‐ray fluorescence spectrometry (XRF) to detect the presence of nonskeletal contaminants in samples of cremains. Eleven samples of cremains were variably combined with concrete mix and analyzed using XRF. Photon counts of elements in each sample were analyzed, and the coefficient of determination (R²) using unweighted linear regression as a function of percent cremains was calculated. Results showed that with changes in the proportion of skeletal material and contaminant, there were significant (R² > 0.90) changes in detected levels of phosphorus, potassium, zinc, aluminum, and sulfur. The use of XRF is concluded to be a valid approach in the identification of the presence of nonskeletal material in potentially contaminated cremains.     

Decaying Mouse Volatiles Perceived by Calliphora vicina Rob.‐Desv.

Volatiles emitted by decaying human remains are in the focus of recent research. The identification of core volatiles in this field is of high importance, because cadaveric volatiles generally show high variation. In this study, the volatile profiles of five mice (Myodes glareolus) were sampled with charcoal filter tubes from their time of death until advanced decay. Eleven compounds were quantitated by means of gas chromatography–mass spectrometry. Electroantennographic experiments with female Calliphora vicina antennae led to the identification of dimethyl trisulfide, dimethyl disulfide, nonanal, hexan‐1‐ol, 1‐octen‐3‐ol, 3‐methylbutan‐1‐ol, and heptanal as electrophysiologically active compounds. When these were compared, dimethyl trisulfide (17 ng/μL) and dimethyl disulfide (11 ng/μL) were found to be emitted in higher concentrations. The roles of these compounds and nonanal as core volatiles for cadaver detection or postmortem time determination and their correlation to the stages of decay and the accumulated degree days are discussed.     

SNP Miniplexes for Individual Identification of Random‐Bred Domestic Cats

Phenotypic and genotypic characteristics of the cat can be obtained from single nucleotide polymorphisms (SNPs) analyses of fur. This study developed miniplexes using SNPs with high discriminating power for random‐bred domestic cats, focusing on individual and phenotypic identification. Seventy‐eight SNPs were investigated using a multiplex PCR followed by a fluorescently labeled single base extension (SBE) technique (SNaPshot^®). The SNP miniplexes were evaluated for reliability, reproducibility, sensitivity, species specificity, detection limitations, and assignment accuracy. Six SNPplexes were developed containing 39 intergenic SNPs and 26 phenotypic SNPs, including a sex identification marker, ZFXY. The combined random match probability (cRMP) was 6.58 × 10^?19 across all Western cat populations and the likelihood ratio was 1.52 × 10¹⁸. These SNPplexes can distinguish individual cats and their phenotypic traits, which could provide insight into crime reconstructions. A SNP database of 237 cats from 13 worldwide populations is now available for forensic applications.     

Development of a PCR High‐Resolution Melt Assay for Artemisia absinthium (Wormwood) and a Triplex Assay with Two Additional “Unregulated Legal High” Species Datura stramonium (Jimson Weed) and Merremia tuberosa (Hawaiian Woodrose),

Artemisia absinthium (wormwood), a common ingredient in absinthe, contains the compound thujone, which is unregulated by the U.S. Drug Enforcement Agency. Thujone can cause an “unregulated legal high” in higher concentrations. The European Union limits thujone from Artemisia species to 35 mg/kg while the U.S. Food and Drug Administration requires less than 10 ppm to be “thujone‐free.” However, individuals can smoke or ingest A. absinthium in different forms. This study developed a polymerase chain reaction (PCR) high‐resolution melt (HRM) assay to detect and identify A. absinthium based on primer specificity, sensitivity, repeatability, and robustness. A triplex assay was performed with three “unregulated legal high” species: Datura stramonium, Merremia tuberosa, and A. absinthium; the PCR HRM assay detected and identified each plant at melt temperatures 77.42 ± 0.20°C, 83.88 ± 0.22°C, and 87.77 ± 0.15°C, respectively. The primer set developed distinguished A. absinthium from a variety of plant species and was successfully triplexed.     

Autolytic changes in blood cells and other tissue cells of human cadavers. I. Viability and ion studies

The viability of white blood cells, spermatozoa of the epididymis, cells from minced spleen, lymph node and lung, and cells aspirated from the bone marrow of the sternum was studied using the vital dye exclusion test. The material comprised 123 medicolegal autopsy cadavers which had been stored in a mortuary cold room at +4 °C up to 10 days after death. Cells which excluded trypan blue were found in various specimens from all cadavers. However, there was marked individual variation in the results. The loss of viability of the white blood cells showed a moderate correlation (r = ?0.78) with the increase in the post-mortem (p.m.) time, whereas the results for other tissues were not so significant. The K⁺ and Mg²⁺ and haemoglobin content and the osmotic resistance of the red cells correlated poorly with the p.m. time. The present results suggest that despite the general assumption that autolytic changes proceed rapidly at the cellular level, individual cells and especially blood cells may remain viable for a long time in cadavers kept at +4 °C.     

Temperature‐dependent Development of Parasarcophaga similis (Meade 1876) and its Significance in Estimating Postmortem Interval

Flesh flies are commonly found insects on decaying corpses that appears slightly later than blowflies, and their development patterns are significant indicators for minimum postmortem interval (PMI_min) estimation. In this study, the flesh fly Parasarcophaga similis (Meade 1876) was reared at nine constant temperatures ranging from 15°C to 35°C to examine indicators for estimating their age. We generated three development models, including isomorphen diagram, isomegalen diagram, and thermal summation model. Larval body length at different rearing temperatures was fit into an L = a + bT + cT² + dT³ equation with which the relationship between the larval body length (L) and the time after larviposition (T) was confirmed. The pupal stage was categorized into 13 substages according to intrapuparial morphological changes, and a detailed table was generated of the pupal developmental stages at five rearing temperatures, 15°C, 20°C, 25°C, 30°C, and 35°C. This study provides fundamental data in supporting P. similis as an indicator for PMI_min estimation.     

Bacterial Cross‐contamination Potential Associated with Contaminated Currency

Previous studies have shown a significant amount of contaminants on paper currencies. It is important to study the transfer of microorganisms between paper currencies to determine whether it meets the level of a human health threat. This cross‐contamination potential was analyzed by seeding new US 1‐dollar bills with Bacillus thuringiensis, and pressing or rubbing them against clean currency to determine the amount of bacteria transfer to the unseeded currency. The transferred amount of bacteria was recovered, plated, incubated, and the colony‐forming units were quantified. Among the recovery methods tested, the most efficient method, vortexing for 10 min with a recovery efficiency of 40 ± 8.1%, was used in this analysis. The resulting transfer rates were 4.8%, 8.6%, and 14.3% when pressed for 24 h, 72 h, and rubbed together, respectively. These transferred amounts of bacteria are significant and have the potential to spread infectious diseases.     

Estimating postmortem interval for human cadavers in a sub‐tropical climate using UV‐Vis‐near‐infrared Spectroscopy

Estimating postmortem interval (PMI) of surface found skeletal remains is challenging. This novel study used UV‐Vis‐NIR spectroscopy to scan soil collected from cadaver decomposition islands (CDIs) ranging from 15‐ to 963‐d postmortem and control soils. A decomposition product spectra model (DPS model) was constructed by deducting the control soil spectra from the CDI soil spectra for the estimation of postmortem indices: PMI (d), ADD₄, ADD₁₀, and ADD₂₀. The DPS model (n = 55) was calibrated and subjected to a full cross‐validation. Calibration R² and RPD for the DPS model ranged from 0.97 to 0.99 and from 6.1 to 9.9, respectively, for the four postmortem interval indices. Validation R² and RPD for the DPS model ranged from 0.73 to 0.80 and from 1.9 to 2.2, respectively. The DPS model estimated postmortem intervals for three test CDIs in a clay soil under perennial grassland (test set 1; n = 3) and six CDIs in a sandy soil under a loblolly pine forest (test set 2; n = 6). Test set 1 had PMI prediction ranges from ?69 to ?117 days, ?796 to +832 ADD₄, +552 to +2672 ADD₁₀, and ?478 to ?20 ADD₂₀ of observed PMI. Test set 2 PMI prediction ranged from ?198 to ?65 days, ?9923 to +2629 ADD₄, ?6724 to +1321 ADD₁₀, and ?2850 to +540 ADD₂₀ of observed PMI. Test set 2 had poor predictions for two CDIs, for all measures of postmortem indices resulting in discussion of sampling depth, effect of body mass index (BMI), and scavenging.     

Potential Use of Hydrocarbons for Aging Lucilia sericata Blowfly Larvae to Establish the Postmortem Interval

Previous studies on Diptera have shown the potential for the use of cuticular hydrocarbons' analysis in the determination of larval age and hence the postmortem interval (PMI) for an associated cadaver. In this work, hydrocarbon compounds, extracted daily until pupation from the cuticle of the blowfly Lucilia sericata (Diptera: Calliphoridae), have been analyzed using gas chromatography–mass spectrometry (GC–MS). The results show distinguishing features within the hydrocarbon profile over the period of the larvae life cycle, with significant chemical changes occurring from the younger larvae to the postfeeding larvae. Further interpretation of the chromatograms using principal component analysis revealed a strong correlation between the magnitudes of particular principal components and time. This outcome suggests that, under the conditions of this study, the cuticular hydrocarbons evolve in a systematic fashion with time, thus supporting the potential for GC–MS analysis as a tool for establishing PMI where such a species is present.     

Alcohol‐Induced Blackout as a Criminal Defense or Mitigating Factor: An Evidence‐Based Review and Admissibility as Scientific Evidence

Alcohol‐related amnesia—alcohol blackout—is a common claim of criminal defendants. The generally held belief is that during an alcohol blackout, other cognitive functioning is severely impaired or absent. The presentation of alcohol blackout as scientific evidence in court requires that the science meets legal reliability standards (Frye, FRE702/Daubert). To determine whether “alcohol blackout” meets these standards, an evidence‐based analysis of published scientific studies was conducted. A total of 26 empirical studies were identified including nine in which an alcohol blackout was induced and directly observed. No objective or scientific method to verify the presence of an alcoholic blackout while it is occurring or to confirm its presence retrospectively was identified. Only short‐term memory is impaired and other cognitive functions—planning, attention, and social skills—are not impaired. Alcoholic blackouts would not appear to meet standards for scientific evidence and should not be admissible.     

Taphonomic Marks on Pig Tissue Due to Cadaveric Coleoptera Activity Under Controlled Conditions

The aim of this work was to study taphonomic marks that cadaveric coleopteran can produce under controlled conditions. To evaluate this, pig trotters were initially exposed to adults of Dermestes maculatus De Geer at 21 ± 5°C and a 12:12‐h day/night cycle. Observations were made and photographs taken every 4–5 days for 9 months. When feeding and reproducing, D. maculatus produced, in both adult and larvae stages, different types of marks such as holes, striations, scratches, and pits in several kinds of tissue such as integumental, connective, and muscular, in both their fresh and dried stages. Bite marks were also evident. The results in this study provide not only taphonomic but also biological and forensic information. This is the first time that this kind of experiment has been performed.     

Field Study on the Attraction and Development of Insects on Human Meconium and Breast‐fed‐Infant Feces

Urogenital myiasis of newborn infants, although rare, is usually considered to indicate neglect due to attraction of flies to feces; however, infant feces have not been determined to attract insects. Human meconium and breast‐fed‐infant feces were used to determine attractiveness to insects and to examine subsequent colonization and growth patterns of insect larvae. Despite small amounts of fecal material present, adults of Lucilia sericata arrived at breast‐fed‐infant feces within five minutes; insects were rarely observed on meconium. Oviposition and growth of L. sericata larvae occurred only on breast‐fed‐infant feces; however, the larvae did not progress beyond the second instar. These data suggest that urogenital myiasis by L. sericata in newborn human infants within the first few days postpartum would not be expected, but desiccation and depletion of infested feces may provide a possible pathway for urogenital myiasis in older newborn infants.     

Effect of Temperature on Six Different Developmental Landmarks within the Pupal Stage of the Forensically Important Blowfly Calliphora vicina (Robineau‐Desvoidy) (Diptera: Calliphoridae)

This study investigates the pupal development times of the blow fly Calliphora vicina, which were studied in the laboratory at six different constant temperatures (15, 20, 23, 25, 28, and 30°C each ± 1°C). Lower thresholds (t_L) for development were estimated from the linear regression of the developmental rates on each temperature. These data have made it possible to calculate the accumulated degree days (ADD) necessary for C. vicina to complete the larval stage and to achieve adult emergence. The minimal duration of development from oviposition to adult emergence was found to be inversely related to temperature. Additionally, six landmarks in pupal development are showed, and for each of the landmarks, the ADD value was calculated for every rearing temperature involved. These data assist in calculating the duration of the pupal stage based on morphological characteristics and would be of great value for future forensic entomological casework.     

Screening Test for Shed Skin Cells by Measuring the Ratio of Human DNA to Staphylococcus epidermidis DNA

A novel screening method for shed skin cells by detecting Staphylococcus epidermidis (S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real‐time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidisDNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples.     

Use of a Gelatin‐based Consolidant to Preserve Thermally‐Altered Skeletal Remains,

Thermally altered skeletal remains can be very fragile and fragmented and are typically further fragmented or even destroyed when handled; recovery of such remains from a scene can therefore be extremely challenging. There are few recommendations and no generally accepted practices for preserving burned bone for recovery and transport. Here, we test whether the application of a gelatin‐based consolidant at the scene can preserve thermally altered bone in the condition and relative anatomical position in which it was discovered. A solution of Knox^® brand gelatin and water was applied to burned pig mandibles using a spray bottle. Qualitative and quantitative analysis indicates that the application of the consolidant significantly decreased fragmentation as compared to untreated controls (p < 0.05), with most of the treated mandibles remaining completely intact after recovery and transport to a secondary location. In addition to the effectiveness for preservation, the method is also easy to apply, inexpensive, and reversible.