Reproducibility of radiographic stage assessment of third molars

The aim of this study was to determine intra- and inter-observer variability of the developing third molar from panoramic radiographs. Formation of third molars was assessed according to stages described by modified Demirjian et al.'s methods: Moorrees et al. [C.F.A. Moorrees, E.A. Fanning, E.E. Hunt, Age variation of formation stages for ten permanent teeth, J. Dent. Res. 42 (1963) 1490-1502] and Solari and Abramovitch [A.C. Solari, K. Abramovitch, The accuracy and precision of third molar development as an indicator of chronological age in Hispanics, J. Forensic Sci. 47 (2002) 531-535]; in addition, data were also analysed unmodified, i.e. Haavikko [K. Haavikko, The formation and alveolar and clinical eruption of the permanent teeth, an orthopantomograph study, Proc. Finn. Dent. Soc. 66 (1970) 104-170] and Demirjian et al. [A. Demirjian, H. Goldstein, J.M. Tanner, A new system of dental age assessment, Hum. Biol. 45 (1973) 211-227]. The sample was a random selection of 73 panoramic radiographs from patients aged 8-24 years. After training, the left maxillary and mandibular third molars were scored on two separate occasions without knowledge of previous scores. Cohen's Kappa and percentage agreement were calculated for each method, for maxillary, for mandibular third molars and combined. Percentage agreement for stages was also calculated. Intra-observer agreement was greater for mandibular third molars compared to maxillary third molars, and better for methods with fewer stages. Kappa values indicated good agreement for most methods; the best was Demirjian et al.'s method for mandibular third molar with very good agreement (K = 0.80) for the first author, good agreement for the second author (K = 0.75) and good agreement between observers (K = 0.75). The stages with best agreement were Demirjian's stage E [A. Demirjian, H. Goldstein, J.M. Tanner, A new system of dental age assessment, Hum. Biol. 45 (1973) 211-227] and Moorrees et al.'s stage Cc and R1/4 [C.F.A. Moorrees, E.A. Fanning, E.E. Hunt, Age variation of formation stages for ten permanent teeth, J. Dent. Res. 42 (1963) 1490-1502]. CONCLUSIONS: Having clearly defined stages and fewer stages allowed better reproducibility of third molar formation.     

Age Estimation Based on Pulp Cavity to Tooth Volume Ratio Using Postmortem Computed Tomography Images

Pulp cavity size is known to decrease with age and can therefore serve as an indicator for age estimation. Here, we evaluated whether reconstructed images of multidetector‐row computed tomography (MDCT) acquired before forensic autopsy are useful for estimating age at death. Images of 136 mandibular first premolars obtained from bodies of known age at death were analyzed, and the volume of the regions corresponding to pulp cavity and that of the whole tooth were determined using a voxel counting function. The pulp cavity was clearly distinguishable from dental hard tissue on the reconstructed images when using a cutoff value of 1400 Hounsfield units. Regression analysis adjusted for sex showed that estimated age correlated significantly with the pulp cavity to tooth volume ratio (r = 0.76). MDCT is gaining more widespread use in forensic medicine, and analyzing dental images to obtain parameters for age prediction is a practical approach for postmortem identification.     

Detection and Identification of Psilocybe cubensis DNA Using a Real‐Time Polymerase Chain Reaction High Resolution Melt Assay,

Psilocybe cubensis, or “magic mushroom,” is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web‐based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy^® Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR^® Green I, Radiant? Green, or LCGreen Plus^® intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR^® Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant? Green dye.     

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.     

Taphonomic Effects of Vulture Scavenging*

Abstract: From July through September 2007, three pig carcasses (Sus scrofa), weighing between 27 and 63 kg were placed outside in a grassy area in central Texas. A surrounding fence prevented entrance by terrestrial scavengers, while allowing avian scavengers unrestricted access. A fourth pig carcass served as a control for the rate of decomposition and was placed in a cage that prevented terrestrial and avian animal access. Modification of the carcasses was recorded through the use of two motion‐sensing digital cameras and daily on‐site observations. American black vultures (Coragyps atratus) and turkey vultures (Cathartes aura) waited c. 24 h before beginning to scavenge and completely skeletonized the carcasses in 3 to 27 h of feeding, leaving scratches on the bones. The accelerated rate of decomposition and the signature markings on the bones should be considered when interpreting taphonomic events and determining an accurate postmortem interval at vulture‐modified scenes.     

Studying survivors of near-lethal suicide attempts as a proxy for completed suicide in prisons

Sex determination is a necessary step in the investigation of unidentified human remains from a forensic context. Teeth, as one of the strongest tissues in the human body, can be used for this purpose. Most studies of sexual dimorphism in teeth are based on the traditional mesiodistal and buccolingual crown measurements. The purpose of this study is to examine the degree of sexual dimorphism in permanent molars of modern Greeks using crown and cervical diagonal diameters, and to evaluate their applicability in sex determination. A total of 344 permanent molars in 107 individuals (53 male and 54 female) from the Athens Collection were examined. Crown and cervical diagonal diameters of both maxillary and mandibular molars were measured. It was found that males have larger molars than females and in 19 out of 24 dimensions measured male molars exceeded female molars significantly (P<0.05). The most dimorphic molars are the maxillary second molar, and the mandibular second and first molars. Although other molars were also sexually dimorphic they did not have a statistically significant difference in all dimensions. Cervical diagonal diameters have found to be more sexually diamorphic than crown diagonal diameters. In discriminant function analysis the variables entered more frequently were the cervical diagonal diameters mainly of mandibular molars. Classification accuracy was found to be 93% for the total sample, 77.4% for upper jaw, and 88.4% for the lower jaw. Accuracy rates were higher for cervical than crown diagonal diameters. The data generated from the present study suggest that this metric method can be useful and reliable for sex determination, especially when the traditional dental measurements are not applicable.     

A Y‐Chromosomal Haplotype with Two Short Tandem Repeat Mutations*

Abstract:  The male‐specific Y‐chromosomal short tandem repeat (STR) is a useful tool in forensic casework. The Y haplotype comprised of 16 loci, which is amplified simultaneously by AmpFlSTR^® Yfiler^TM PCR kit and provides strong exculpatory evidence in individual identification. We reported a rare Y‐STR profile with a null allele at the DYS448 locus and an off‐ladder allele at the DYS456 locus, when genotyping material from a vaginal swab in an alleged rape case. Sequence analysis revealed that the DYS448 null allele was a true type of null allele because of a total deletion of 11 upstream repeats and 9 bp of the N₄₂ region, and there were numerous primer binding site mutations as well. The amplicon of the DYS456 locus was a small 92‐bp fragment that was off‐ladder, and sequencing analysis showed that there were only 10 repeats (AGAT)₁₀. This Y chromosome haplotype that was comprised of two variations provided helpful evidence for personal identification.     

Color Stability of Dental Restorative Materials Submitted to Heat Sources,for Forensic Purposes

During postmortem examination of the dental arches of carbonized victims, dental restorative materials may be found. The aim of this study was to evaluate the effect of heat source action on the color stability of composite resin (CR) and glass ionomer cement (GIC) restorations, to discriminate between them and compare with antemortem dental data. Sixty bovine teeth (30 CR and 30 GIC) were prepared (6 × 6 × 2 mm) and separated into groups (n = 10). The color readouts were taken by spectrophotometer, before and after heat action (100°C, 200°C, 300°C), in an oven for 15 min. There were color alterations for all coordinates (ΔE, ΔL*, Δa* eΔb*) for both materials. GIC presented greater change. The authors concluded that it is possible to distinguish between the materials by the color changes analyzed by instrumental method, helping victim identification.     

Pyrolysis Products of Linear Alkylbenzenes— Implications in Fire Debris Analysis*

Abstract: In this case report, potential interferences from an improvised fire‐extinguishing agent, a dishwashing liquid, containing linear alkylbenzene sulfonates (LAS), was studied. The presence of linear alkylbenzenes (LABs) in the fire debris sample was identified from the summed ion profile (SIP) analysis. It was found that the LAS from dishwashing liquids produce LABs by thermal degradation. Direct pyrolysis of a LAS‐containing dishwashing liquid at 300°C yielded a distribution of LABs in the SIP. LABs began to break down at pyrolysis temperatures between 450 and 500°C and completely break down by 800°C. Observed pyrolysis breakdown products of LABs included toluene, ethylbenzene, meta‐, para‐, and ortho‐xylenes, propylbenzene, indane, naphthalene, and 1‐ and 2‐methylnaphthalenes. These data suggested the presence of LABs in fire debris evidence might complicate subsequent analysis because their breakdown products contained some of the target compounds common to ignitable liquid identification. Therefore, a positive determination of the presence of foreign ignitable liquids should be carefully evaluated when there is a presence of LABs in the SIP.     

Age estimation of adults from dental radiographs

Previous studies have shown that with advancing age the size of the dental pulp cavity is reduced as a result of secondary dentine deposit, so that measurements of this reduction can be used as an indicator of age. The aim of the present study was to find a method which could be used to estimate the chronological age of an adult from measurements of the size of the pulp on full mouth dental radiographs. The material consisted of periapical radiographs from 100 dental patients who had attended the clinics of the Dental Faculty in Oslo. The radiographs of six types of teeth from each jaw were measured: maxillary central and lateral incisors and second premolars, and mandibular lateral incisors, canines and first premolars. To compensate for differences in magnification and angulation on the radiographs, the following ratios were calculated: pulp/root length, pulp/tooth length, tooth/root length and pulp/root width at three different levels. Statistical analyses showed that Pearson's correlation coefficient between age and the different ratios for each type of tooth was significant, except for the ratio between tooth and root length, which was, therefore, excluded from further analysis. Principal component analyses were performed on all ratios, followed by regression analyses with age as dependent variable and the principal components as independent variables. The principal component analyses showed that only the two first of them had significant influence on age, and a good and easily calculated approximation to the first component was found to be the mean of all the ratios. A good approximation to the second principal component was found to be the difference between the mean of two width ratios and the mean of two length ratios, and these approximations of the first and second principal components were chosen as predictors in regression analyses with age as the dependent variable. The coefficient of determination (r²) for the estimation was strongest when the ratios of the six teeth were included (r² = 0.76) and weakest when measurements from the mandibular canines alone were included (r² = 0.56). Measurement on dental radiographs may be a non-invasive technique for estimating the age of adults, both living and dead, in forensic work and in archaeological studies, but the method ought to be tested on an independent sample.     

Comparison of Decomposition Rates and Faunal Colonization of Carrion in Indoor and Outdoor Environments

Abstract: Decomposition and insect colonization of pig (Sus scrofa L.) carcasses were observed over a 42‐day period inside and outside a house in a suburban region of Edmonton, Alberta. Three freshly killed pig carcasses were placed outdoors on grass and three carcasses were placed in separate rooms inside a house in a suburban residential area. The carcasses were examined and sampled regularly. Outside carcasses were colonized rapidly by Calliphora vicina (R‐D), Lucilia sericata (Meigen), Lucilia illustris (Meigen), Eucalliphora latifrons (Hough), Cynomya cadaverina (R‐D), Phormia regina (Meigen), and Protophormia terraenovae (R‐D). There was a delay of 5 days before inside carcasses were first colonized but all species except E. latifrons and L. illustris readily colonized at this time. Many more insects colonized the outside carcasses, and these were rapidly skeletonized. Inside, much fewer insects were present and decomposition was slowed and colonization extended. Dispersal patterns of postfeeding larvae inside the house on hard substrate were greatly expanded compared with those from carcasses on grass.     

Dental Age Estimation: A Test of the Reliability of Correctly Identifying a Subject Over 18 Years of Age Using the Gold Standard of Chronological Age as the Comparator

This study was designed to test the reliability of using the third molars to demarcate between child and adult status. A total of 2000 dental panoramic tomographs were used for assessment of the calculated age using the 8‐stage system of tooth development and applied to all four third molars. The LL8 was also assessed using this 8‐stage system. For each tooth development stage, the Normal distribution and percentile summary data were estimated. The calculated dental age compared with the chronological age was statistically significantly different (p < 0.001) for both females and males giving underestimates of the true age. Comparison of single tooth dental age and chronological age was only slightly different. The most important finding is that the assignment to above or below the 18‐year threshold, in the age range 17 years to 19 years, could be wrong on up to 50% of occasions.     

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.     

Mitochondrial DNA Extraction from Burial Soil Samples at Incremental Distances: A Preliminary Study

Preservation variance of soil DNA is neglected in the literature, and exceptional cases exaggerate amplification capabilities. This study sought to amplify a short mitochondrial fragment (212 bp) specific to Sus scrofa domesticus from the soil surrounding decomposing pig remains from an open‐air locale. Samples collected above the body at incremental distances after 145 days of initial placement yielded pig DNA. A secondary sampling was collected in 2017, approximately 768 days after burial. Inhibition tests corroborated that pig DNA was no longer present in the soil resulting in a loss of original DNA between 145 and 768 days. The results provide evidence that genetic material leaches out radially from the source and DNA fragments longer than 200 bp do not persist in soil for a relatively short timeframe in western Montana. The conclusions support the collection of soil in crime scene investigation procedures within the first few months of decomposition.     

Avian Scavenging of Small‐Sized Pig Carcasses in Central Florida: Utilizing GIS to Analyze Site Variables Affecting Skeletal Dispersal

Scavengers can significantly alter a forensic scene and consume, modify, disarticulate, and disperse bodies on the ground surface. The research purpose was to examine vulture scavenging in central Florida, USA. Four small‐sized pig (Sus scrofa) carcasses were left on the ground surface of two microenvironments (shaded and open) at a secure site with game cameras. Dispersal data were mapped and analyzed using geographical information systems spatial analysis digital mapping tools. The primary avian scavengers recorded included black vultures (Coragyps atratus) and turkey vultures (Cathartes aura), as well as bald eagles (Haliaeetus leucocephalus). Carcass dispersal patterns were impacted by foliage density (grass height and concentrations of bushes and trees) and proximity to the perimeter fence. While the majority of skeletal elements were dispersed within 6 m of the carcass deposition locations, dispersion occurred over a greater distance in the wooded microenvironment. Overall, vulture behaviors deleteriously destroyed and changed the context of the scene, with black vultures having the greatest impact.     

Arthropods of Forensic Importance on Pig Carrion in the Coahuilan Semidesert,Mexico

Abstract: This is the first report of an ongoing research to establish a sarcosaprophagous arthropod database in the Coahuilan semidesert. Seven pigs (Sus scrofa L.) were used as human models to determine succession in an open urban area during the 2007 winter–spring period. Arthropods were collected manually and from pitfall traps. Carcass biomass loss, as well as arthropod colonization, was recorded during 71 days postmortem. Five decomposition stages were identified during which most abundant orders were found to be Diptera, Coleoptera, and Hymenoptera. Lucilia sericata (Meigen), Chrysomya rufifacies (Macquart), Necrobia rufipes (DeGeer), Dermestes maculatus (DeGeer), Pheidole hyatti Emery, and Pogonomyrmex rugosus Emery stood out as dominant species.     

Reliable and Robust Molecular Sexing of the Hen Harrier (Circus cyaneus) Using PCR‐RFLP Analysis of the CHD 1 Gene

The hen harrier (Circus cyaneus) is a bird of prey that is persecuted in the United Kingdom, and there is a need for a DNA‐based individual identification and sexing system for the use in forensic investigations. This study reports a new set of PCR primers for the chromo‐helicase‐DNA‐binding protein 1 gene, which allows sexing using PCR‐RFLP. Instead of exonic primers that amplify across a large intron, this set consists of a primer within the intron, enabling reduction in amplicon sizes from 356 to 212 bp and 565 to 219 bp in W and Z chromosomes. DNA degradation and dilution experiments demonstrate that this set is significantly more robust than one that amplifies across the intron, and sequencing of the intronic primer‐binding region across several individuals shows that it is highly conserved. While our objective is to incorporate this primer set into an STR‐based individualization kit, it may in the meantime prove useful in forensic or conservation studies.     

A Checklist of Calliphoridae Blowflies (Insecta,Diptera) Associated with a Pig Carrion in Central Brazil*

Abstract: Although the Cerrado is the second major Brazilian biome, few studies have been undertaken about its entomofauna. Blowflies have an important role in forensic entomology, helping in the determination of postmortem intervals. The main goal of this exploratory study was to identify and to catalog the blowfly species associated with a pig carcass. The study was conducted in a pasture in Brasília, Distrito Federal. A pig (Sus scrofa) was killed with a .22 caliber shot in the frontal region of the head. Adult blowflies were surveyed daily from June 1 through 30, 2004. A total of 14,910 adult calliphorids were collected, representing eight species: Chrysomya albiceps, C. megacephala, Cochliomyia macellaria, Chloroprocta idioidea, Hemilucilia semidiaphana, H. segmentaria, Lucilia cuprina, and L. eximia. C. albiceps was the most frequent species, amounting to 94.76% of the catch. Five decomposition stages were observed, and for calliphorids, the most attractive stage was the bloated one.     

Vertical Lip Position and Thickness in Facial Reconstruction: A Validation of Commonly Used Methods for Predicting the Position and Size of Lips

This study examined several methods used to estimate oral fissure position, lip margin position, and lip thickness recommended by Angel, George, Lebedinskaya, Taylor, Wilkinson et al., Balueva and Veselovskaya. A sample of 86 lateral head cephalograms of adult subjects from central Europe were measured and the actual and predicted dimensions were compared. The best estimation for oral fissure position was “opposite the lower ¾ mark of maxillary incisors” (error of 1.3 mm). Upper lip margin was predicted best by “upper ¼ mark of maxillary incisors” (error of 1.7 mm), and lower lip margin by “cementum‐enamel junction of mandibular incisors” (error of 2.3 mm). The regression equations of Wilkinson et al. displayed least error (1.3 mm and 1.8 mm, respectively) for upper and lower lip thickness, and method of George (error of 3.4 mm) for total lip thickness.