Use of laser-scanning confocal microscopy in the detection of diagenesis in bone.

This research demonstrates the value of laser scanning confocal microscopy (LSCM) as a research tool in osteological studies, and diagenetic studies in particular. LSCM combines properties of light and scanning electron microscopy using laser light to excite fluorophores throughout the z-axis, developing a 3-D image. Using differential staining and selecting for specific wavelengths of light, one can image targeted materials. This research is divided into two parts: visualizing bone structures such as proteins and their decompositional products and visualizing diagenesis. Part one of this study utilized pig bones as a means of testing the overall ability of LSCM to fluoresce bone. Twenty-three samples were imaged, including 13 samples from a decompositional study conducted 5 years previous, and 10 "fresh" samples collected from a commercial butcher. This part of the study determined that protein and organic components of the bone could be fluoresced and diagenetic alteration could be imaged. The second part of the study used human samples as a means of imaging and mapping diagenetic alterations. The second part of the study used 13 samples, including 4 clinical, 7 ancient, and 2 modern controls. The pig study used Basic Fuchsin and SlowFade Gold stains, while the human study used toluidine blue. Images were also taken with unstained elements. The results of the non-human study found that a fresh bone fluoresced differently than that of a 5-year subset, while the results of the human study confirmed these findings and determined that the bone diagenesis can be mapped using LSCM.

Reprint of: Breaking the binary: The identification of trans-women in forensic anthropology.

In the cases of transgender and gender non-conforming people, a description of their physical remains, including biological sex, may have little correlation with their social identity, delaying and often confusing the issue of identification. Some transgender individuals have sought to alter their physical appearance in order to better reflect their gender. One group of surgical modifications for trans-women, or individuals transitioning from MTF (male-to female), is known as facial feminization surgery (FFS) which involves the reduction and contour of the forehead, chin and jaw contour, and rhinoplasty, to give trans-women smoother, smaller facial features. The purpose of this research is to examine the impact of FFS on measurement-based methods of cranial sex assessment, such as discriminant function analysis. The goal is to develop guidelines for correctly recognizing and supporting the identification of trans-women. The results of this study demonstrate that evidence of gender can be found in the facial skeletons of MTF transgender individuals who have undergone facial feminization surgery, and that forensic anthropologists should consider individuals who do not fit into the traditional sex binary when assessing the sex of unidentified skeletal remains.

Breaking the binary: The identification of trans-women in forensic anthropology.

In the cases of transgender and gender non-conforming people, a description of their physical remains, including biological sex, may have little correlation with their social identity, delaying and often confusing the issue of identification. Some transgender individuals have sought to alter their physical appearance in order to better reflect their gender. One group of surgical modifications for trans-women, or individuals transitioning from MTF (male-to female), is known as facial feminization surgery (FFS) which involves the reduction and contour of the forehead, chin and jaw contour, and rhinoplasty to give trans-women smoother, smaller facial features. The purpose of this research is to examine the impact of FFS on measurement-based methods of cranial sex assessment, such as discriminant function analysis. The goal is to develop guidelines for correctly recognizing and supporting the identification of trans-women. The results of this study demonstrate that evidence of gender can be found in the facial skeletons of MTF transgender individuals who have undergone facial feminization surgery, and that forensic anthropologists should consider individuals who do not fit into the traditional sex binary when assessing the sex of unidentified skeletal remains.

Resolving Reflection and Resolution in 3D Imaging of Fresh Bone.

Structured light scanning is a noninvasive, accurate, and cost-effective 3D imaging technique, but due to reflection issues is yet to be utilized for tool mark analysis on fresh bone. During imaging, reflection from shiny surfaces, such as greasy bone, disrupts image formation. This study tested the David SLS-1 scanner's ability to image saw marks and explored six strategies to reduce reflection by [1] dulling the surface or [2] altering the projected light. The surface was dulled by freezing, talcum powder, dulling spray, or compressed air. The projected light was altered with a diffuser or limited to single pattern-coded. Results demonstrated that the resolution was insufficient for capturing minute details of striae. All six tests failed to reduce reflection sufficiently to produce complete images, but projecting vertical pattern-coded light showed the most promise. Future research is required concentrating on enhancing resolution and exploring the role of pattern-coded light in reducing reflection.

An Evaluation of the Calce Method for Age Estimation.

The Calce method of skeletal age estimation (Am J Phys Anthropol, 148, 2012 and 11) uses the acetabular surface of the os coxa and was developed using 90 individuals from the J.C.B. Grant Skeletal collection. From this collection, pilot tests using a combined sample size of 55 randomized individuals yielded an accuracy of 54.5%. To eliminate the possible issue of variation within the collection, 30 individuals from those that Calce specifically used were assessed by two analysts. Accuracies of 53.3% and 56.7% were obtained, compared with Calce's reported accuracy of 81% (Am J Phys Anthropol, 148, 2012 and 11). This study also used 30 Japanese individuals from the Nagasaki University modern cadaver collection. Due to the high interobserver error (43.3%) and the low accuracies achieved (40% and 46.7%), the Calce method does not perform well on Japanese samples. The low accuracy of this method in general suggests that the trait descriptions should be refined to assist analysts in properly utilizing the method.

Evaluation of age estimation technique: testing traits of the acetabulum to estimate age at death in adult males.

This study evaluates the accuracy and precision of a skeletal age estimation method, using the acetabulum of 100 male ossa coxae from the Grant Collection (GRO) at the University of Toronto, Canada. Age at death was obtained using Bayesian inference and a computational application (IDADE2) that requires a reference population, close in geographic and temporal distribution to the target case, to calibrate age ranges from scores generated by the technique. The inaccuracy of this method is 8 years. The direction of bias indicates the acetabulum technique tends to underestimate age. The categories 46-65 and 76-90 years exhibit the smallest inaccuracy (0.2), suggesting that this method may be appropriate for individuals over 40 years. Eighty-three percent of age estimates were ±12 years of known age; 79% were ±10 years of known age; and 62% were ±5 years of known age. Identifying a suitable reference population is the most significant limitation of this technique for forensic applications.

Personal identification using the frontal sinus.

The frontal sinuses are known to be unique to each individual; however, no one has tested the independence of the frontal sinus traits to see if probability analysis through trait combination is a viable method of identifying an individual using the frontal sinuses. This research examines the feasibility of probability trait combination, based on criteria recommended in the literature, and examines two other methods of identification using the frontal sinuses: discrete trait combinations and superimposition pattern matching. This research finds that most sinus traits are dependent upon one another and thus cannot be used in probability combinations. When looking at traits that are independent, this research finds that metric methods are too fraught with potential errors to be useful. Discrete trait combinations do not have a high enough discriminating power to be useful. Only superimposition pattern matching is an effective method of identifying an individual using the frontal sinuses.

Soft tissue decomposition of submerged, dismembered pig limbs enclosed in plastic bags.

This study examines underwater soft tissue decomposition of dismembered pig limbs deposited in polyethylene plastic bags. The research evaluates the level of influence that disposal method has on underwater decomposition processes and details observations specific to this scenario. To our knowledge, no other study has yet investigated decomposing, dismembered, and enclosed remains in water environments. The total sample size consisted of 120 dismembered pig limbs, divided into a subsample of 30 pig limbs per recovery period (34 and 71 days) for each treatment. The two treatments simulated non-enclosed and plastic enclosed disposal methods in a water context. The remains were completely submerged in Lake Ontario for 34 and 71 days. In both recovery periods, the non-enclosed samples lost soft tissue to a significantly greater extent than their plastic enclosed counterparts. Disposal of remains in plastic bags therefore results in preservation, most likely caused by bacterial inhibition and reduced oxygen levels.

The effects of soil environment on postmortem interval: a macroscopic analysis.

Burial environment, in particular soil moisture, has a significant impact on the type, rate, and extent of bone degradation, which ultimately affects estimations of the postmortem interval (PMI). The purpose of this research is to determine the effects of soil moisture on the color, weight, condition, and texture of bone as it relates to the PMI. Bone changes occurring over two different time intervals (2 and 5 months) were examined using 120 sus scrofa leg bones. During each time interval bones were buried in two soil environments, one of which was drier than the other. The bones in both environments lost weight over time but the net weight loss was greater for bones in the higher moisture environment. There was no change in color, texture, or overall condition, indicating that 150 days is not long enough for such alterations to occur, regardless of the moisture level of the burial environment.

Reconstructing the sequence of events surrounding body disposition based on color staining of bone*.

Literature regarding bone color is limited to determining location of primary and secondary dispositions. This research is the first to use bone color to interpret the sequence of events surrounding body disposition. Two scenarios were compared-bones buried and then exposed on the ground surface and bones exposed then buried. Forty juvenile pig humeri with minimal tissue were used in each scenario with an additional 20 controls to determine if decomposing tissue affects bone color. Munsell Color Charts were used to record bone color of surface and 2.5 cm cross-sections. Results reveal five main surface colors attributed to soil, sun, hemolysis, decomposition, and fungi. Fungi on buried bones suggests prior surface exposure. Cross-sections of strictly buried bones are identical to buried then exposed bone, stressing the importance of bone surface analysis. Cross-sectioning may help verify remains have been exposed then buried. Decomposition of excess tissue creates minimal color staining.

Sex determination of adolescent skeletons using the distal humerus.

Accurate determination of the sex of immature skeletal remains is difficult in the absence of DNA, due to the fact that most sexually dimorphic features of the human skeleton develop as secondary sex characteristics during adolescence. Methods of assessment of adult skeletons cannot reliably be applied to adolescent skeletons because of the transitional nature of the skeleton at puberty and the variability of the adolescent growth spurt. The purpose of this work was to evaluate the accuracy of Rogers's method of morphological sex determination using the distal humerus (Rogers: J Forensic Sci 44 (1999) 55-59) to assess the sex of adolescent skeletons. The sample consists of 7 documented adolescent skeletons from the Christ Church Spitalfields collection at the British Museum of Natural History and 35 from the Luis Lopes skeletal collection housed in the National History Museum (Museu Bocage) of the University of Lisbon, Portugal. Ages range from 11 to 20 years. The technique achieved an accuracy of 81% on the combined sample of 42. This method can be applied to adolescent skeletons once the trochlea begins fusing to the humeral diaphysis, which occurred by age 11 years in the test samples.

Bone weathering patterns of metatarsal v. femur and the postmortem interval in Southern Ontario.

Twenty-five defleshed pig femora and 25 metatarsals were placed outdoors and observed over 291 days to establish: (1) bone weathering patterns for use in estimating time since death in Southern Ontario and (2) whether larger (femora) or smaller (metatarsals) bones provide a better indicator of time since death. Pig hind limbs were observed to determine a timeline for decomposition of soft tissues during the fall and winter. Ambient air temperature, humidity, precipitation, sunlight, soil pH, and freezing and thawing were considered as factors affecting the breakdown of bone. Weathering patterns were observed based on the extent of bleaching, amount of periosteum and soft tissues present, as well as the appearance of greasiness, cracking and flaking of cortical bone. Both entomological activity and climatic conditions affected soft tissue decomposition. Animal activity affected both the process of bone weathering and soft tissue decomposition, causing variability in sample decomposition and bone breakdown. The variation in microenvironment, partially caused by soil composition, introduced variability in bone weathering rates. Four bone weathering stages were established based on patterns observed. Femora proved to be more resilient and showed more degrees of change due to weathering, thus proving to be a better indicator of time since death than metatarsals.

Taphonomic changes to blunt force trauma: a preliminary study.

This study examines the effects of taphonomic processes on blunt force trauma (BFT) through an experimental study involving pig heads. Of particular concern is the possibility that taphonomic changes can create pseudo-trauma and/or conceal evidence of actual trauma. BFT was inflicted on 10 pig skulls using a hammer. The skulls were subsequently exposed to the environment for 12 months. Seven taphonomic changes were evaluated: the freeze-thaw cycle; rodent gnawing; carnivore scavenging; presence/weight of soil; presence/weight of rain and snow; movement/displacement of bones; and discoloration due to sun bleaching and grass staining. Taphonomic effects varied between cancellous, compact, fresh, and degreased bone. Freezing and thawing, exposure to rain and snow, movement of the skulls, and soil erosion altered and, in some cases disguised, pre-existing trauma. Rodent and carnivore activity did not obliterate evidence of BFT. Recommendations for evaluating BFT on remains affected by taphonomic processes are presented. As each taphonomic process outlined by this study has the potential to disguise antemortem injury, the authors propose that one must carefully examine large, circular openings in the skull that may represent the remnant evidence of BFT.

Variation in cortical bone histology within the human femur and its impact on estimating age at death.

Histological methods for the estimation of age at death using cortical bone are based on the evaluation of microstructural changes over time. Since histological analysis is a destructive method, most techniques attempt to limit the amount of cortical bone needed for analysis. Sample location, however, can have a significant effect on the accuracy of these methods. Furthermore, research demonstrates that both intersection and intrasection variation is present at the midshaft of the femur, which is the primary location for estimating age at death in humans. This research determines the extent of regional variation within the adult human femur and its effect on age estimation. Secondary osteon lamellae and Haversian canal ratio and cortical thickness were quantified. Thompson's All Males Left Femur regression equation was used to estimate age. Results show that significant regional variation occurs in the estimated ages derived from the posterior aspect of the femoral shaft and significant intrasection variation occurs in age estimates from the mid and mid-distal cross-sections. Thus, the inter and intrasection variation that occurs in bone remodeling within the femoral cortex has the potential to produce significant differences amongst age estimates taken from various femoral diaphyseal locations compared to the age estimated from the standard location used in Thompson's core method (1978). The results indicate that the use of this histological method is dependant on the ability to correctly identify the four anatomical locations, but the extracted core used for age estimation is not necessarily confined to the anterior midshaft.

Determining the sex of human remains through cranial morphology.

Sex determination is the keystone of a biological profile, yet few qualitative methods of cranial sex determination have been tested. This analysis examines the accuracy and precision of 17 morphological features of the skull commonly used to determine the sex of unknown skeletal remains. The sample consists of 46 identified skulls from the 19th century St. Thomas' Anglican Church Cemetery in Belleville, Canada. Nasal aperature, zygomatic extension, malar size/rugosity, and supraorbital ridge proved the most useful; of secondary value are chin form and nuchal crest; mastoid size is of tertiary consideration; nasal size and mandibular symphysis/ramus size rank fourth; forehead shape ranks fifth; and palate size/shape are sixth. Skull size/architecture provides an internal standard to assess the relative sizes of other traits. This research is a necessary step in establishing the credibility of morphological sex determination with respect to the Daubert and Mohan criteria for admissibility in a court of law.

Recognition of cemetery remains in a forensic context.

The purpose of this paper is to provide guidelines for the recognition of human remains from modem and historic cemeteries found in a forensic context. Three avenues of evidence may be pursued to confirm the origin of cemetery remains: context, condition of the body, and associated artifacts. This article outlines types of North American cemeteries, demonstrating how land use over time has resulted in many being closed, moved, or forgotten, leaving only the context to indicate their presence. The condition of human cemetery remains varies considerably depending on cultural practices and burial environment, but many exhibit combinations of the following traits: dried or embalmed tissue; erosion of bony pressure points; cortical bone flaking; and bone damage due to autopsy or embalming. Examples of artifact types useful in recognizing cemetery remains are also provided. Two cases from British Columbia, Canada are presented to demonstrate the diagnostic features of a disturbed cemetery burial.

Expert testimony and positive identification of human remains through cranial suture patterns.

North American forensic anthropological research should conform to the Daubert criteria (U.S.A.) and Mohan ruling (Canada) to ensure admissibility in a court of law. Positive identification through radiographic comparison of antemortem and postmortem cranial suture patterns was evaluated in light of these criteria. The technique is based on reliable principles, but problems with terminology and the resolution of radiographs make Sekharan's method difficult to apply. Using the location, length, and slope of a suture's component lines, rather than Sekharan's descriptions of sutural configurations, it is possible to determine the probability of a particular suture pattern occurring in more than one individual. A match of four consecutive lines is sufficient to establish positive identification. This approach meets the Daubert and Mohan criteria, although resolution of radiographs is still a major limitation. Computed tomography (CT) scans may prove a more useful modality for positive identification, due to better resolution and greater availability.

Crime scene ethics: souvenirs, teaching material, and artifacts.

Police and forensic specialists are ethically obliged to preserve the integrity of their investigations and their agencies' reputations. The American Academy of Forensic Sciences and the Canadian Society of Forensic Science provide no guidelines for crime scene ethics, or the retention of items from former crime scenes. Guidelines are necessary to define acceptable behavior relating to removing, keeping, or selling artifacts, souvenirs, or teaching specimens from former crime scenes, where such activities are not illegal, to prevent potential conflicts of interest and the appearance of impropriety. Proposed guidelines permit the retention of objects with educational value, provided they are not of significance to the case, they are not removed until the scene is released, permission has been obtained from the property owner and police investigator, and the item has no significant monetary value. Permission is necessary even if objects appear discarded, or are not typically regarded as property, e.g., animal bones.

Identifying chop marks on cremated bone: a preliminary study.

The purpose of this analysis is to evaluate the effects of burning on hacking trauma inflicted with a cleaver and to assess the diagnostic potential of cleaver marks exposed to fire. Thirty pig forelimbs (radius and ulna) and 30 beef ribs were each subjected to five blows with a cleaver and five cuts with a knife prior to burning in an outdoor fire. Bones were deliberately agitated to ensure maximum cremation and induce fragmentation. Results indicate that hacking weakens bone, making fire-induced fragmentation more likely at the sites of trauma. Chop marks were easily identified on burned bone, their characteristics largely unaffected by cremation.