DNA typing of human remains found in damp environments

DNA typing is often used to determine identity from human remains. The environment to which the material has been exposed, however, is crucial for the success of the investigation. Damp conditions in particular can cause a rapid degradation of DNA, even in bone and teeth, and thus reduce the chances of successful typing. Here, we present the results of investigations performed on four skulls that had been lying in a damp environment for periods ranging from almost 1 year to about 45 years. In none of these cases was DNA typing successful on bone or, where present, on teeth. Where remnants of brain tissue were used, however, complete STR typing was possible in one case, partial STR typing in another, and mtDNA sequencing could be carried out in three cases. These findings suggest that brain tissue is relatively resistant to putrefaction in damp environments and, unlike bone, appears to exhibit a certain degree of protection against DNA degradation.     

Collecting touch DNA from glass surfaces using different sampling solutions and volumes: Immediate and storage effects on genetic STR analysis

Touch DNA has become increasingly important evidence in todays' forensic casework. However, due to its invisible nature and typically minute amounts of DNA, the collection of biological material from touched objects remains a particular challenge that underscores the importance of the best collection methods for maximum recovery efficiency. So far, swabs moistened with water are often utilized in forensic crime scene investigations for touch DNA sampling, even though an aqueous solution provokes osmosis, endangering the cell's integrity. The aim of the research presented here was to systematically determine whether DNA recovery from touched glass items can be significantly increased by varying swabbing solutions and volumes compared with water-moistened swabs and dry swabbing. A second objective was to investigate the possible effects of storage of swab solutions prior to genetic analysis on DNA yield and profile quality when stored for 3 and 12 months, as is often the case with crime scene samples. Overall, the results indicate that adapting volumes of the sampling solutions had no significant effect on DNA yield, while the detergent-based solutions performed better than water and dry removal, with the SDS reagent yielding statistically significant results. Further, stored samples showed an increase in degradation indices for all solutions tested, but no deterioration in DNA content and profile quality, allowing for unrestricted processing of touch DNA samples stored for at least 12 months. One further finding was a strong intraindividual change in DNA amounts observed over the 23 deposition days which may be related to the donor's menstrual cycle.     

Typing of deoxyribonucleic acid (DNA) extracted from compact bone from human remains.

The application of deoxyribonucleic acid (DNA) typing methods for the potential identification of unknown human remains was investigated. DNA was isolated from compact bone tissue from badly decomposed bodies and from known and unknown human remains, using a decalcification and ion wash procedure. Restriction fragment length polymorphism (RFLP) analysis of variable number of tandem repeats (VNTR) loci yielded results in some cases, but more often the DNA was too degraded to produce RFLP patterns. No RFLP profiles could be obtained from putrefied soft tissues. However, DNA extracted from compact bone tissue of human remains up to eleven years old was successfully amplified using the polymerase chain reaction (PCR) for the VNTR loci D1S80, D17S5, COL2A1, and APO B, as well as the HLA-DQ alpha locus. This is especially significant, since PCR results were obtained from those samples whose DNA had been degraded substantially and had yielded no RFLP patterns. All DNA types determined from the compact bone tissue from decomposed bodies whose identification had been established first by other means (and whose parents or offspring were available for typing) demonstrated mendelian inheritance of the alleles of the loci analyzed. These results suggest that amplification and typing of DNA extracted from compact bone of human remains could be useful in establishing the identity of a person, as well as in excluding possible false identifications.     

Comparison of DNA yield after long-term storage of Second World War bone samples

Sample storage is of paramount importance in forensic genetics laboratories since only optimal storage enables successful recovery of DNA from old bones that contain very low amount of severely degraded DNA. When identification of missing persons from skeletal remains is completed, bone sample is routinely stored at -20 °C for long-term storage for retesting in future, if necessary. After molecular genetic analyses of Slovenian Second World War (WWII) victims, small fragments of femurs were stored at -20 °C. Reduction in DNA recovery has been observed in frozen liquid DNA extracts by some authors and the goal of our study was to explore how freezing of bone samples affects the preservation of DNA. To achieve this goal, the difference in DNA yield in extracts obtained from WWII bones analyzed in 2009 (data from published paper) and DNA yield in extracts obtained from the same bones (piece sampled next to the one used in 2009) taken out of the freezer after long-term storage on -20 °C for 10 years was examined, using the same extraction method and the same quantification kit. Up to 100 ng DNA/g of bone powder was obtained from 57 WWII femurs and up to 31 ng DNA/g of bone powder from the same femurs investigated after long-term storage in this study. 0,5 g of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 device (Qiagen) and DNA quantity determined with the Human Quantifiler kit (TFS). Statistical analysis showed significant difference in DNA yield in extracts obtained from WWII bones in 2009 and extracts obtained from the same bones stored at -20 °C after 10 years. As reported for frozen liquid DNA extracts, reduction in recovery of DNA was confirmed for frozen bone samples as well.     

Interdisciplinary cooperation of forensic sciences--results of a simulation exercise

In a workshop held on the occasion of the foundation of the INFW, the Interdisciplinary Network of Forensic Sciences (www.infw.org), 14 scientists from 10 fields of expertise were allotted to 3 teams. Each team had to independently solve the same fictitious forensic case. In this, several corpses or skeletons in varying degrees of decomposition were found in the remains of an old bunker during a large building project. After a set time limit of 45 minutes, the approaches to the task were noted on individual flipcharts by each team. A comparison of the solutions suggested by the three very heterogeneously composed teams revealed a high degree of similarity. However, particularly the "exotic" disciplines developed surprising approaches. The experiment was an interesting and instructive experience for all participants and underscores the necessity of interdisciplinary cooperation in solving complex forensic questions.     

White‐tailed Deer as a Taphonomic Agent: Photographic Evidence of White‐tailed Deer Gnawing on Human Bone

Ungulate gnawing on bone has been reported in the taphonomic and zooarchaeological literature, but there are no known reports of ungulates altering human remains. Herein, we report on the first known photographic evidence of deer gnawing human remains. As described in nonhuman scavenging literature, forking of the bone characterizes the taphonomic effect of deer gnawing in this case, which is distinct from the effect caused by other scavengers. This type of osteophagia during the winter season is consistent with previously documented behavior of deer gnawing on nonhuman bone, possibly to obtain minerals absent in their diet. In this study, we briefly discuss the distinguishing features of ungulate gnawing, the reasons for this behavior, and possible confusion with other common types of scavenging and modification. This report contributes to taphonomic literature covering the range of animal interactions with human skeletal remains.     

A review of the current understanding of burned bone as a source of DNA for human identification

Identification of incinerated human remains may rely on genetic analysis of burned bone which can prove far more challenging than fresh tissues. Severe thermal insult results in the destruction or denaturation of DNA in soft tissues, however genetic material may be preserved in the skeletal tissues. Considerations for DNA retrieval from these samples include low levels of exogenous DNA, the dense, mineralised nature of bone, and the presence of contamination, and qPCR inhibitors. This review collates current knowledge in three areas relating to optimising DNA recovery from burned bone: 1) impact of burning on bone and subsequent effects on sample collection, 2) difficulties of preparing burned samples for DNA extraction, and 3) protocols for bone decalcification and DNA extraction. Bone decalcification and various DNA extraction protocols have been tested and optimised for ancient bone, suggesting that prolonged EDTA (Ethylenediaminetetraacetic acid) demineralisation followed by solid-phased silica-based extraction techniques provide the greatest DNA yield. However, there is significantly less literature exploring the optimal protocol for incinerated bones. Although burned bone, like ancient and diagenetic bone, can be considered “low-copy”, the taphonomic processes occurring are likely different. As techniques developed for ancient samples are tailored to deal with bone that has been altered in a particular way, it is important to understand if burned bone undergoes similar or different changes. Currently the effects of burning on bone and the DNA within it is not fully understood. Future research should focus on increasing our understanding of the effects of heat on bone and on comparing the outcome of various DNA extraction protocols for these tissues.     

Analysis of artificial radiocarbon in different skeletal and dental tissue types to evaluate date of death

Radiocarbon dating, with special reference to the modern bomb-curve, can provide useful information to elucidate the date of death of skeletonized human remains. Interpretation can be enhanced with analysis of different types of tissues within a single skeleton because of the known variability of formation times and remodeling rates. Analysis of radiocarbon content of teeth, especially the enamel in tooth crowns, provides information about the date of formation in the childhood years and in consideration of the known timing of tooth formation can be used to estimate the birth date after 1950 ad. Radiocarbon analysis of modern cortical and trabecular bone samples from the same skeleton may allow proper placement on the pre-1963 or post-1963 sides of the bomb-curve as most trabecular bone generally undergoes more rapid remodeling than does most cortical bone. Pre-1963 bone formation would produce higher radiocarbon values for most trabecular bone than for most cortical bone. This relationship is reversed for formation after 1963. Radiocarbon analysis was conducted in this study on dental, cortical, and trabecular bone samples from two adult individuals of known birth (1925 and 1926) and death dates (1995 and 1959). As expected, the dental results correspond to prebomb-curve values reflecting conditions during the childhoods of the individuals. The radiocarbon content of most bone samples reflected the higher modern bomb-curve values. Within the bone sample analyses, the values of the trabecular bone were higher than those of cortical bone and supported the known placement on the pre-1963 side of the bomb-curve.     

Colonization of diatoms in and on porcine bone substrate and considerations of diatom ecology for forensic science

The presence of diatom algae in bone marrow has been used as forensic evidence of drowning for several decades; however, these studies are based on known or suspected recent drowning events. This study addresses the potential for diatoms to enter the bone marrow of skeletal remains, that is, de-fleshed long bones post-mortem. In laboratory and field experiments, bones were either inflicted with two access points by a cut and acid pitting or left intact. The bones were submerged in water for at least 1 week and up to 3 months. Samples of the bone surface and marrow were inspected for diatoms. The analysis considered the time required for diatoms to enter marrow and whether genus characteristics like size or mobility affect entry. The presence of an access point influenced diatom entry in that bones without an introduced access point had zero to one diatom present in the marrow, whereas a bone with an access point had >150 diatoms present in the marrow. The results of both laboratory and field phases suggest that diatoms will reliably colonize bone in as quickly as 1 week, establishing and maintaining communities for at least 3 months. However, the bone surface assemblages differ from the source community. Bone marrow displayed even more restrictive access to diatom colonization, resulting in communities dominated by small raphid diatoms. Based on these findings, we suggest some caveats on the use of diatoms as trace evidence in forensic science with recommendations for future avenues of research.     

Identification of Missing Norwegian World War II Soldiers,in Karelia Russia

This article presents the multidisciplinary effort in trying to identify the skeletal remains of 100 Norwegian soldiers serving in the German army, killed in Karelia Russia in 1944, from the recovery of the remains through the final identification using DNA. Of the 150 bone samples sent for DNA testing, 93 DNA profiles were obtained relating to 57 unique individuals. The relatives could not be directly contacted as the soldiers were considered as traitors to Norway; therefore, only 45 reference samples, relating to 42 cases of the missing, were donated. DNA matches for 14 soldiers and 12 additional body part re‐associations for these individuals were found. Another 24 bone samples were re‐associated with 16 individuals, but no familial match was found. More than six decades after the end of WWII, DNA analysis can significantly contribute to the identification of the remains.     

Multi-agent scavenging patterns in Hawai‘i: A forensic archaeological and skeletal case study

Knowledge of the behavior of local fauna can aid forensic investigators in developing awareness of site formation processes. In Hawai‘i, little has been published on the effects of feral domestic pig (Sus scrofa) and feral domestic dog (Canis familiaris) scavenging and bone dispersal on field recovery and laboratory observations. In this Pacific tropical setting, the most consequential terrestrial taphonomic agents are pigs and dogs, both in terms of hard tissue modification and dispersal of remains across the landscape. In 2017, an archaeologist discovered the remains of an unidentified decedent on the island of Kauaʻi, State of Hawai‘i during a cultural resource management survey. Subsequently, a forensic recovery team in conjunction with Kaua‘i police and crime scene investigators used archaeological techniques, including pedestrian survey, tape-and-compass, and GPS mapping, to map and recover the remains. A feral pig trail transected various areas of the recovery site and corresponded with the distribution pattern of recovered skeletal material, including both the main concentration more broadly dispersed skeletal elements. While much of the skeleton was present, missing or unrecovered skeletal elements are consistent with expectations based on existing literature. Much of the postmortem bone deformations were characteristic of marks related to feral dog and/or feral pig scavenging. These results assisted local investigators in deciding the manner of death, as well as providing the family with an accounting of the decedent’s remains for burial. Thus, forensic anthropologists and archaeologists need to understand and develop knowledge of local animal behavior to recover and interpret human remains of medicolegal significance.     

An investigation of model forensic bone in soil environments studied using infrared spectroscopy

Infrared spectroscopy has been used to examine changes to bone chemistry as a result of soil burial. Pig carcasses were buried as part of a controlled field study, and pig bone was used in soil environments established in the laboratory. The variables of species type, bone pretreatment, soil type and pH, moisture content, temperature, and burial time were investigated. The crystallinity index (CI) and the organic and carbonate contents of the bones were monitored. The data revealed decreasing trends in the organic and carbonate contents and an increase in the CI of the bone with burial time. An acidic soil environment and soil type are the factors that have the most influence on bone chemistry as a result of burial. The study demonstrates the potential of infrared spectroscopy as a straightforward method of monitoring the changes associated with aging of bones in a variety of soil environments.     

Effect of Temperature on Bone Tissue: Histological Changes

The analysis of burned human remains has been of great interest among forensic anthropologists largely due to the difficulty that their recovery, classification, reconstruction, and identification present. The main purpose of this analysis is to present histological methodology for the interpretation of bones altered by thermal processes. We include analyses of the microscopic changes among bones exposed to different temperatures, with the goal of establishing categories of histological morphology in relation to fire temperature. Samples of bone (ilium) were exposed systematically to controlled temperatures. Analysis of the resulting histological changes has allowed the formation of a clear four‐stage classification of the alterations observed. This classification should prove useful in assessing bone changes in relation to temperature of exposure, particularly in cases where this temperature was previously not known.     

Use of aquatic insects in determining submersion interval

Although its potential is great, the use of aquatic insects in determining submersion intervals at death-scene investigations has not been exploited in the past. Aquatic environments have no known true specific indicator species, as do terrestrial habitats. However, aquatic environmental studies show that organisms may colonize a substrate dependent on factors such as size, position, exposure to current, water temperature, current speed, water depth, the presence of algal communities, or detritus. Certain aquatic insects such as the chironomid midges (Diptera, Chironomidae), and the caddisflies (Trichoptera), are capable of colonizing immersed bodies; and with the known biology of a specific species of insect for a certain geographic area, time intervals of submersion can be established.     

Degradation pathways of 4-methylmethcathinone in alkaline solution and stability of methcathinone analogs in various pH solutions

Analogs of methcathinone (MC), a psychoactive stimulant, are in circulation all over the world. These analogs have been assumed to be unstable in alkaline solutions, as is MC itself. The aims of this study were: (i) to identify the degradation products of 4-methylmethcathinone (4-MMC), a typical MC analog, in solution at pH 12 and to determine the degradation pathway, (ii) to investigate the effects of antioxidants such as l-ascorbic acid and sodium sulfite on the degradation of 4-MMC, and (iii) to investigate the stability of seven MC analogs (4-MMC, 4-, 3-, or 2-fluoromethcathinone, 4-methoxymethcathinone, N-ethylcathinone, and N,N-dimethylcathinone) in solutions at different pHs.1-(4-Methylphenyl)-1,2-propanedione (MPPD), 4-methylbenzoic acid (MBA), N,4-dimethylbenzamide (DMBA), and N-acetyl-4-MMC (N-Ac-4-MMC) were identified as the degradation products of 4-MMC in pH 12 solution by gas chromatography-mass spectrometry. There are two degradation pathways for 4-MMC as follows: (a) 4-MMC→MPPD→MBA→DMBA and (b) 4-MMC→N-Ac-4-MMC. Oxidants such as dissolved oxygen were presumed to be involved in this degradation based on the suppressive effects generated by the addition of antioxidants. All of the seven MC analogs tested were stable in acidic (pH 4) solution but degraded in neutral-to-basic solutions. Their degradation rates increased with increasing pH, and varied with their chemical structures. These findings will be very useful for not only forensic analysis but also future pharmacokinetic analysis.     

Preliminary results of an investigation on postmortem variations in human skeletal mass of buried bones

Extreme fragmentation can complicate the inventory of human skeletal remains. In such cases, skeletal mass can provide information regarding skeleton completeness and the minimum number of individuals. For that purpose, several references for skeletal mass can be used to establish comparisons and draw inferences regarding those parameters. However, little is known about the feasibility of establishing comparisons between inherently different materials, as is the case of curated reference skeletal collections and human remains recovered from forensic and archaeological settings. The objective of this paper was to investigate the effect of inhumation, weather and heat exposure on the skeletal mass of two different bone types. This was investigated on a sample of 30 human bone fragments (14 trabecular bones and 16 compact bones) that was experimentally buried for two years after being submitted to one of four different heat treatments (left unburned; 500?°C; 900?°C; 1000?°C). Bones were exhumed periodically to assess time-related mass variation. Skeletal mass varied substantially, decreasing and increasing in accordance to the interchanging dry and wet seasons. However, trends were not the same for the two bone types and the four temperature thresholds. The reason for this appears to be related to water absorption and to the differential heat-induced changes in bone microporosity, volume, and composition. Our results suggest that mass comparisons against published references should be performed only after the skeletal remains have been preemptively dried from exogenous water.     

Selective culturing and genus-specific PCR detection for identification of Aeromonas in tissue samples to assist the medico-legal diagnosis of death by drowning

The detection of autochthonous aquatic bacteria in tissue samples from drowning cases is increasingly considered as an alternative approach to assist the medico-legal diagnosis of death by drowning. Bacteria belonging to the genus Aeromonas may be suitable candidates for this application as they are ubiquitous in natural aquatic environments but are generally not part of the human microbiota. The research aims of this study were (i) to develop a sensitive, specific and rapid screening and confirmation method for Aeromonas species in tissue samples and (ii) to evaluate aseptic sternal puncture as a post-mortem sample technique and bone marrow as an alternative matrix to provide evidence of death by drowning. The presence of Aeromonas in tissue samples was verified by cultivation using the selective media Ampicillin Dextrin Agar (ADA) and Ryan's Aeromonas Medium. The use of ADA medium was found most optimal for the sensitive, inexpensive and quick detection of aeromonads in human tissue samples. Positive culture plates were confirmed by harvesting all colonies for DNA extraction and subsequent PCR amplification using Aeromonas genus-specific primers. Aeromonads were detected in lung swab, blood and bone marrow of drowned bodies (n=3), but were negative in these three matrices for all negative controls (n=90) tested. Bone marrow proved to be a suitable alternative matrix and can be sampled post-mortem by an aseptic sternal puncture. In conclusion, this study confirms previous indications that aeromonads in cultures from blood of water bodies can be considered a potential marker for drowning. Given the fact that the number of immersed bodies (drowned and non-drowned) included in this study is statistically not significant, however, more tissue samples need to be investigated to confirm the validity of these methods to aid the diagnosis of death by wet drowning.     

Effect of 1,2-indanedione on PCR-STR typing of fingerprints deposited on thermal and carbonless paper

The determination of the date of death from bone remains is of scientific interest but also has important legal implications. The establishment of the postmortem interval (PMI) is a very complex problem because of the great number of intrinsic factors that may alter the normal course of postmortem change, such as the age, sex, constitution and previous physiological and pathological states of the subject, and external factors. In order to evaluate the utility of X-ray diffraction and the measurement of some components in dating bone remains, a total of 69 long bones from 69 different cadavers (41 males, 28 females) with a mean age of 68 years (S.D.=17.6, range 12-97) were used. The bones were removed from cement tombs of Murcia Cemetery, where they had lain for documented times of between 7 and 54 years (S.D.=11.6, mean time 17.6 years). We have studied potassium, sulphur, nitrogen, urea, total protein, phosphorus, and some X-ray diffraction (XRD) parameters related to the degree of crystallinity of the mineral component in medullar and cortical bone zones to establish which of the two provides the most useful information for calculating the PMI. In the overall analysis of our data, we believe that the use of both XRD and biochemical analyses (especially of urea, potassium and sulphur) particularly in the cortical zone of the bone could be an alternative method for dating osseous remains.     

The Difficult Task of Diagnosing Prostate Cancer Metastases on Dry Bone

The interpretation of pathology on skeletal remains is mandatory for implementing the biological profile and for disease recognition. Prostate cancer is one of the most common tumors, with a high preference for the skeleton as a primary site of metastasis. Its diagnosis on bone is however still ambiguous, due to its “osteoblastic” and resorptive manifestation. This study investigates distribution and appearance of prostate cancer metastases on dry bone on six known cases (selected from the Milano Cemetery Skeletal Collection) and one healthy individual. A macroscopic inspection was performed highlighting the abnormalities observed, describing location, shape, dimension, and aspect. A great amount of proliferative and mixed lesions was noticed, but also cases of pure lytic lesions were displayed. The multiple appearances of the manifestations observed display the difficulty in correctly identifying such a pathology, but also the potential and advantages provided by investigating a study sample with known antemortem history.