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.     

Potential Use of Bacterial Community Succession in Decaying Human Bone for Estimating Postmortem Interval,,

Bacteria are taphonomic agents of human decomposition, potentially useful for estimating postmortem interval (PMI) in late‐stage decomposition. Bone samples from 12 individuals and three soil samples were analyzed to assess the effects of decomposition and advancing time on bacterial communities. Results indicated that partially skeletonized remains maintained a presence of bacteria associated with the human gut, whereas bacterial composition of dry skeletal remains maintained a community profile similar to soil communities. Variation in the UniFrac distances was significantly greater between groups than within groups (p < 0.001) for the unweighted metric and not the weighted metric. The members of the bacterial communities were more similar within than between decomposition stages. The oligotrophic environment of bone relative to soft tissue and the physical protection of organic substrates may preclude bacterial blooms during the first years of skeletonization. Therefore, community membership (unweighted) may be better for estimating PMI from skeletonized remains than community structure (weighted).     

DNA Identification of Commingled Human Remains from the Cemetery Relocated by Flooding in Central Bosnia and Herzegovina

The floods in Bosnia and Herzegovina in May 2014 caused landslides all over the country. In the small village of ?eri?i, near the town of Zenica, a landslide destroyed the local cemetery, relocated graves, and commingled skeletal remains. As the use of other physical methods of identification (facial recognition, fingerprint analysis, dental analysis, etc.) was not possible, DNA analysis was applied. DNA was isolated from 20 skeletal remains (bone and tooth samples) and six reference samples (blood from living relatives) and amplified using PowerPlex^® Fusion and PowerPlex^®Y23 kits. DNA profiles were generated for all reference samples and 17 skeletal remains. A statistical analysis (calculation of paternity, maternity, and sibling indexes and matching probabilities) resulted in 10 positive identifications. In this study, 5 individuals were identified based on one reference sample. This has once again demonstrated the significance of DNA analysis in resolving the most complicated cases, such as the identification of commingled human skeletal remains.     

The Use of an Alternate Light Source for Detecting Bones Underwater

When searching underwater crime scenes or disaster scenes for fragmentary human remains, it may be advantageous for forensic divers to be able to detect the presence of bones and teeth among other marine materials (such as shells and rocks). In terrestrial environments, this can typically be accomplished by visual and instrumental methods, but underwater conditions make it difficult to employ detection and sorting techniques in these environments. This study investigates fluorescence of bones and teeth and other marine materials using a submersible alternate light source (ALS) and concludes that an ALS can be a useful tool for detecting bones and teeth in underwater searches as well in terrestrial searches and laboratory environments. The results could impact the methods and equipment used by forensic divers and forensic anthropologists when searching for skeletal remains, potentially increasing the quantity and efficiency of forensic evidence recovered.     

The Use of Femoral Neck Axis Length to Estimate Sex and Ancestry

Having multiple reliable methods of estimating sex and ancestry from various skeletal features increases the likelihood of identifying skeletal remains. Femoral neck axis length (FNAL), as measured in living individuals, has been shown to vary by sex and ancestry. FNAL has not, however, been previously measured directly from skeletonized remains and investigated for its potential use in forensic anthropological applications. This research proposes a method for measuring FNAL from skeletal remains, determines the reliability and repeatability of the measurement, and assesses the validity of FNAL in sex and ancestry estimation. Results showed low interobserver error in the measurement of FNAL (TEM = 0.33 mm, R = 0.99). Significant differences in FNAL were found between sexes as well as between American Black, American White, and Native American groups. FNAL can correctly classify sex in ~86% of all cases and is considered valuable to sex estimation. The value of FNAL to ancestry estimation, however, is considered limited.     

Citrate Content of Bone for Time Since Death Estimation: Results from Burials with Different Physical Characteristics and Known PMI

A recently introduced method to determine the postmortem interval (PMI) based on quantification of the citrate content in bone was applied on the temporal bones and femora of 20 individuals buried in wooden coffins (WO) and body bags (BB), respectively. Concerning known vs. calculated PMI, a significant difference between the temporal and the femur bone samples of the same individuals was observed in the BB group (p = 0.012). In contrast, differences were insignificant for the WO group (p = 0.400). Moreover, similar levels of underestimation of PMIs resulted from the analysis of the femora for both burial groups (p = 0.247). Also, there was consistently less citrate preserved in the flat temporal bones as compared to the femora, indicating that the cortical layer of the long bones should be preferentially employed for citrate‐based PMI estimations. The results call for additional research on subsurface‐buried and surface‐deposited remains to enhance the accuracy of the published PMI equation.     

Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically Important Chrysomya rufifacies (Diptera: Calliphoridae),

Entomological protocols for aging blowfly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analyzing fly larvae differ, most rely on light microscopy, genetic analysis, or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically important blowfly Chrysomya rufifacies using high‐content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti‐ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, nonbleaching, autofluorescence of larval posterior spiracles. High‐content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC.     

DNA Typing for the Identification of Old Skeletal Remains from Korean War Victims*

Abstract: The identification of missing casualties of the Korean War (1950–1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA‐matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y‐chromosomal STR, and mtDNA‐genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini‐ and Y‐STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains.     

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.     

An Experimental Study of Vertebrate Scavenging Behavior in a Northwest European Woodland Context

Vertebrate scavengers can modify surface deposited human remains which can hinder forensic investigations. The effects of such scavenging vary between species and regions. Published research into the effects of the scavenging of human remains is dominated by work from North America with few studies covering Northwestern Europe. Forensic scientists, investigators, and police search officers in Northwestern Europe are often left questioning on a basic level as to which scavengers are active and how they might affect human remains. This paper presents the results of a field study utilizing deer (Cervus nippon; Capreolus capreolus) as surface deposits observed by motion detection cameras in a British woodland. The most common avian and rodent scavenger species recorded included the buzzard (Buteo buteo), carrion crow (Corvus corone), wood mouse (Apodemus sylvaticus), and gray squirrel (Sciurus carolinensis). The scavenging behaviors observed were affected by seasonality, rates of decomposition, and insect activity. Scavenging by buzzards, unlike carrion crows, was most frequent during fall to winter and prior to insect activity. Overall, avian scavengers modified and scavenged soft tissue. Rodents scavenged both fresh and skeletonized remains with gray squirrels only scavenging skeletal remains. Wood mice were most active in winter and scavenged both soft tissue and bone.     

A Method for Estimating Sex Using the Clavicle,Humerus, Radius,and Ulna

Sex estimation from skeletal remains can be an important part of preliminary identification. The best source of information for estimating sex is the pelvis but it is not always available for analysis. For these cases, a probabilistic sex estimation method is presented using combinations of standard and alternative measurements of the clavicle, humerus, radius, and ulna. Various equations are developed that are not population specific and that are applicable in various recovery scenarios. The equations were tested using four independent samples (n > 370), including a forensic sample. Allocation accuracies vary by test sample and equation and are consistently good (87.4–97.5%) except for a sample of very small males that show the extreme effects of poverty and mortality bias. For many of the cases where allocation was incorrect, the probabilistic approach indicated that no confidence should be placed in the incorrect allocation and the unknown should be classified as sex indeterminate.     

Cranial Nonmetric Variation and Estimating Ancestry*

Abstract: Historically, when predicting the ancestry of human skeletal remains, forensic anthropologists have not fully considered the variation within human populations, but instead have relied on a typological, experience‐based approach. Unfortunately, reliance on observer experience has produced a method that is as much an art as it is a science. This research focuses on the frequency distribution and inter‐trait correlations of 11 common morphoscopic traits to demonstrate that the experience‐based approach to ancestry prediction is indeed an art that is unscientific, because it is unreplicable, unreliable, and invalid. Ten of 11 traits examined had frequency distributions with significant differences (p < 0.001) between groups, but the range in variation of these traits far exceeds previous assumptions. Such within group variation clearly demonstrates that extreme trait expressions are not reliable for estimating ancestry through visual observation alone, but instead that these traits should be analyzed within a statistical framework.     

Metric Assessment of the Pubic Bone Using Known and Novel Data Points for Sex Estimation,

Biological sex estimation of skeletal remains is essential in forensic and archaeological analyses. Anthropologists most often use the pelvis, which is the most sexually dimorphic element both morphologically and metrically. While nonmetric pubic bone features have been studied extensively, few metric studies have examined this individual bone for dimorphism. For this study, three observers examined three previously identified and ten novel measurements of the pubic body on a modern sample of isolated pubic bones from the Maricopa County Forensic Science Center (FSC), in Phoenix, Arizona (n = 400). A relationship between pubic body measurements and biological sex was demonstrated, with significant correlations. Discriminant function analyses found that five measurements, four of which were novel, discriminated between males (89%) and females (86%). Observer experience level did not significantly impact the results. These five measurements were reliable and show promise for inclusion in metric methods for assessment of sex.     

Weight References for Burned Human Skeletal Remains from Portuguese Samples

Weight is often one of the few recoverable data when analyzing human cremains but references are still rare, especially for European populations. Mean weights for skeletal remains were thus documented for Portuguese modern cremations of both recently deceased individuals and dry skeletons, and the effect of age, sex, and the intensity of combustion was investigated using both multivariate and univariate statistics. The cremains from fresh cadavers were significantly heavier than the ones from dry skeletons regardless of sex and age cohort (p < 0.001 to p = 0.003). As expected, males were heavier than females and age had a powerful effect in female skeletal weight. The effect of the intensity of combustion in cremains weight was unclear. These weight references may, in some cases, help estimating the minimum number of individuals, the completeness of the skeletal assemblage, and the sex of an unknown individual.     

Bioelectrical Impedance as a Technique for Estimating Postmortem Interval,,

Estimation of postmortem interval (PMI) is a critical component of death investigation. A cadaver can be hypothesized to be a resistor–capacitor (RC) circuit the impedance (Z) of which changes in a quantifiable manner as the cadaver decomposes. This hypothesis was tested using bioelectrical impedance analysis (BIA) equipment to apply a current with a fixed amplitude at a single frequency to four cadavers over time and measuring two components of Z, resistance (R) and reactance (X_c). Quadratic regression analysis between Z and accumulated degree days (ADD) showed a statistically significant parabolic relationship. The parabolic relationship poses an initial challenge to the use of the method, and additional research is needed to address this issue. However, the results of the reported research support the hypothesis that Z measured using BIA has a relationship to PMI.     

A Reliable Regression‐Based Approach for Reassociating Human Skeletal Elements of the Lower Limbs from Commingled Assemblages

Accurate sorting of commingled human remains comprises a fundamental requirement for all further anthropological analyses. The lower limb bones are particularly important for reconstructing biological profiles. This study introduces a metric technique for sorting these elements using eight standard anthropological measurements and 222 adult individuals from Greece. The bones utilized were the os coxae, the femora, the tibiae and the tali. Simple regression analyses were used to develop functions for reassociating articulating bones, providing strong correlations (r = 0.74–0.95, p‐value <0.05) and high coefficients of determination (r²=0.54–0.91). Blind tests demonstrated that combining metric and morphoscopic techniques provides an excellent sorting accuracy for the hip and knee joints (ten of ten individuals), allowing for a reliable reassociation between a sex and age indicator (os coxae) and a body size indicator (femur). Overall, these results indicate the high value of metric methods in sorting commingled human remains.     

Scavenger Species-typical Alteration to Bone: Using Bite Mark Dimensions to Identify Scavengers

Scavenger-induced alteration to bone occurs while scavengers access soft tissue and during the scattering and re-scavenging of skeletal remains. Using bite mark, dimensional data to assist in the more accurate identification of a scavenger can improve interpretations of trauma and enhance search and recovery methods. This study analyzed bite marks produced on both dry and fresh surface deposited remains by wild and captive red fox (Vulpes vulpes) and Eurasian badger (Meles meles), as well as domestic dog (Canis familiaris). The bite marks produced by foxes were distinguishable from those made by badgers and dogs based on ranges of mean length and breadth of pits. The dimensional data of bite marks produced by badgers and dogs were less discernible. Bone modifications vary due to a variety of factors which must be considered, such as scavenger species-typical scavenging behavior, scavenger species' dentition, condition and deposition of remains, and environmental factors.     

Healed Depressed Parasagittal Skull Fractures—A Feature of Archaic Australian Aboriginal Remains

The skeletal remains of eight Australian Aboriginals with healed depressed skull fractures were examined. Male:female ratio 5:3; age range 20‐60 yrs. Burial dates by ¹⁴C dating in three cases were 500 years BP (n = 2) and 1300 BP. There were 13 healed depressed skull fractures manifested by shallow indentations of cortical bone and thinning of diploe, with no significant disturbance of the inner skull tables. Nine (69%) were located within 35 mm of the sagittal suture/midline. These lesions represent another acquired feature that might be helpful in suggesting that a skull is from a tribal Aboriginal individual and may be particularly useful if the remains are represented by only fragments of calvarium. While obviously not a finding specific to this population, these healed injuries would be consistent with the possible results of certain types of conflict behavior reported in traditional Aboriginal groups that involved formalized inflicted blunt head trauma.     

Epiphyseal Union of the Cervical Vertebral Centra: Its Relationship to Skeletal Age and Maturation of Thoracic Vertebral Centra

Epiphyseal union stages for cervical vertebral centra (ring epiphyses) were documented for 55 individuals (females and males, ages 14–27 years) from the Terry Collection, using the Albert and Maples method 1, to examine both its relationship to age at death and to thoracic data collected from the same individuals using the same method. Results showed a moderate correlation between cervical ring union and age (r = 0.63, p = 0.000), and a fairly low correlation between cervical and thoracic ring union (r = 0.41, p = 0.002). Paired samples t‐tests yielded a statistically significant difference between cervical and thoracic union mean values (p = 0.01). Union progressed earlier in cervical vertebrae and in females. Results indicated fairly substantial variation in both sexes. Findings may serve as a basic guideline for estimating a general age range at death for unknown skeletal remains and to corroborate findings from other skeletal age indicators.     

Correlation of Molecular Expression with Diel Rhythm of Oviposition in Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) and Implications for Forensic Entomology

This study explored the molecular mechanisms potentially underlying blow fly nocturnal oviposition. A behavioral study revealed that Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) possesses a diel rhythm of oviposition in light under 12:12 light/dark conditions. Reversal to 12:12 dark/light resulted in oviposition behavior changing to align with the adjusted regime in most females, but four of 59 experimental females lacked a diel rhythm of oviposition (were arrhythmic). Real-time PCR was used to monitor the molecular expression levels of known circadian genes per and tim in C. vicina to determine whether gene expression and behavior correlated. As with behavior, reversing light/dark conditions changed rhythmic gene expression to align with an adjusted light regime. This suggests that although it is unlikely that C. vicina will colonize dead bodies at night, arrhythmic females and oviposition in the dark was demonstrated.