Frozen human bone: a microscopic investigation

The taphonomic effects of prolonged extreme cold and freezing on human bone have received little research attention. Questions of specific interest include whether previously frozen bone can be identified and whether freezing alters the structural integrity enough to prevent histological aging. There is no evidence from previous studies that freezing damages the structural integrity, and to date no research investigating the freezing process on bone microstructure has been undertaken. This research attempts to distinguish histologically previously frozen bone from nonfrozen bone by identifying patterned defects. To determine the effects of freezing in bone microstructure using light and scanning electron microscopy (SEM), several human bone sections were subjected to prolonged freezing and allowed to thaw before thin sectioning. Light microscopy failed to demonstrate statistically significant differences between frozen and nonfrozen specimens. SEM analysis revealed fractures, although these lacked pattern and did not occur systematically throughout the section. Evidence of microstructural changes caused by liquid expansion, however, was remarkable but did not alter the structural integrity of the microstructure. The results of this study suggest that freezing does not alter the process of histomorphological analysis.     

An Examination of the Transition of Fracture Characteristics in Long Bones from Fresh to Dry in Central Florida: Evaluating the Timing of Injury

It is important to conduct timing of injury research analyzing fracture characteristics at known postmortem intervals (PMI) because bone can retain fresh characteristics throughout the PMI. Defleshed pig (Sus scrofa) long bones were fractured weekly in two environments (full sun and shade) over 14 weeks in Central Florida and fracture characteristics were categorized (N = 136) for analysis. Results of analysis of variance (ANOVA) using time in weeks (PMI) as a dependent variable indicate significant relationships between PMI and Fracture Angle (p < 0.001), Fracture Surface (p < 0.001), and Fracture Outline (p < 0.001). Fracture characteristics associated with perimortem trauma (smooth Fracture Surfaces and curved or V‐shaped Fracture Outlines) were commonly observed. Analysis of fracture characteristics for each environment demonstrated similar patterns. Overall, the loss of only fresh fracture characteristics for each bone was noted earlier in the PMI for the Central Florida region than previously reported.     

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).     

The Effect of Time on Bone Fluorescence: Implications for Using Alternate Light Sources to Search for Skeletal Remains

Bones fluoresce when exposed to certain wavelengths of shortwave light, and this property can be useful in locating and sorting skeletal remains in forensic contexts. The proteins in bone collagen are largely responsible for its fluorescent properties, but these proteins degrade and denature over time. This study examined the fluorescence of bones from four temporal groups (recent, semi‐recent, ancient, and historic) ranging from 0 to 1064 years before present. Specimens were photographed under 490 nm wavelength light, and fluorescence was quantified by converting intensity to a gray scale value based on the RGB color model using ImageJ^® software. Significant (p < 0.05) differences were found in mean fluorescence between all four temporal groups, and a 0.324 coefficient of correlation indicates a significant (inverse) relationship between fluorescence and time. Bone fluorescence decreases with time, but some fluorescence is retained even in older samples. Fluorescence can therefore be reliably used in many modern skeletal remains searches.     

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.     

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.     

Egg freezing and egg banking: empowerment and alienation in assisted?reproduction

With the development of rapid freezing of human oocytes, many programs have reported IVF success rates comparable to those achieved with fresh eggs and thawed frozen embryos. Egg freezing is now gaining professional and regulatory acceptance as a safe and effective technique for women who wish to avoid discarding excess embryos, who face fertility-threatening medical treatments, or who want to preserve their eggs for use when they are better situated to have a family. This article focuses on the uses of and justification for egg freezing, the path to professional acceptance, the variability in success rates, and the controversy over freezing eggs for social rather than medical reasons. It also addresses the emergence of egg banking as a separate sector in the infertility industry, the regulatory issues that it poses, and its effect on egg donation. Key here is the legal control of stored eggs by banking women and their options when they wish to dispose of those eggs. The analysis is framed around empowerment and alienation. Egg freezing is generally empowering for women, but the donation or sale of unused eggs to infertile women, egg bankers, and researchers also raises issues of alienation.     

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.     

Radiodense bullet wipe around osseous entrance gunshot wounds

“Bullet wipe” is the material deposited by a bullet on any surface with which it comes into contact after it is fired and may contain debris from the gun barrel, including particles of primer and metal fragments from previously fired bullets. X‐ray analysis is a non‐destructive method by which traces of metallic elements can be visually detected. The analysis of osseous defects for radiodense bullet wipe (RBW) assists in determining the presence or absence of perforating gunshot wounds, especially in fragmented, skeletonized remains. The aim of our current study was to determine the frequency of RBW around entrance firearms injuries that perforated bone. We prospectively analyzed entrance gunshot wounds for RBW over a three‐year period using digital X‐ray analysis (n = 59). We retrospectively reviewed the corresponding autopsy reports to determine the frequency of RBW by biologic sex, reported ancestry, age‐at‐death, location of wound, manner of death, range of fire, bullet caliber, and presence of bullet jacket. Data were analyzed by Fisher's exact test or Chi‐square test with significance levels accepted at p < 0.05. RBW was present in 66% (n = 39) of examined cases. Decedent characteristics did not significantly alter RBW distribution, including biologic sex (p = 0.75), reported ancestry (p = 0.49), and age‐at‐death (p = 0.43). Additionally, the location of the osseous entrance gunshot wound, manner of death, range of fire, and cartridge caliber did not affect RBW detection. All cases involving non‐jacketed rounds (n = 5) showed RBW (p = 0.30). To our knowledge, this study is the first to report the frequency of RBW detection from osseous entrance gunshot wounds.     

Bone Mineral Density Adult Age Estimation in Forensic Anthropology: A Test of the DXAGE Application

Estimating age‐at‐death of individuals represented only by skeletonized human remains is a fundamental aspect of forensic anthropological casework. Recently, several researchers have proposed that bone mineral density (BMD) is a useful predictor of age‐at‐death in forensic contexts. Navega et al. (JFS 63(2):497–503) developed an online application called DXAGE for calculating age‐at‐death from BMD parameters. This study tests the utility of DXAGE by utilizing data from the National Health and Nutrition Examination Survey (NHANES). BMD data from a female subsample (n = 470) of the NHANES 2007–2008 dataset were analyzed, and the relationship between predicted age and real age was examined. Inaccuracy was 14.25 years, and bias was ?7.20 years. Results show that there is a weak correlation between predicted and actual age (r = 0.47) using the DXAGE application. While BMD data are potentially useful for predicting age age‐at‐death, the DXAGE application should be used cautiously in forensic anthropological contexts.     

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.     

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.     

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.     

The Effects of Soil Texture on the Ability of Human Remains Detection Dogs to Detect Buried Human Remains

Despite technological advances, human remains detection (HRD) dogs still remain one of the best tools for locating clandestine graves. However, soil texture may affect the escape of decomposition gases and therefore the effectiveness of HDR dogs. Six nationally credentialed HRD dogs (three HRD only and three cross‐trained) were evaluated on novel buried human remains in contrasting soils, a clayey and a sandy soil. Search time and accuracy were compared for the clayey soil and sandy soil to assess odor location difficulty. Sandy soil (p < 0.001) yielded significantly faster trained response times, but no significant differences were found in performance accuracy between soil textures or training method. Results indicate soil texture may be significant factor in odor detection difficulty. Prior knowledge of soil texture and moisture may be useful for search management and planning. Appropriate adjustments to search segment sizes, sweep widths and search time allotment depending on soil texture may optimize successful detection.     

A New Method for Determination of Postmortem Interval: Citrate Content of Bone*

Abstract: Few accurate methods exist currently to determine the time since death (postmortem interval, PMI) of skeletonized human remains found at crime scenes. Citrate is present as a constituent of living human and animal cortical bone at very uniform initial concentration (2.0 ± 0.1 wt %). In skeletal remains found in open landscape settings (whether buried or not), the concentration of citrate remains constant for a period of about 4 weeks, after which it decreases linearly as a function of log(time). The upper limit of the dating range is about 100 years. The precision of determination decreases slightly with age. The rate of decrease appears to be independent of temperature or rainfall but drops to zero for storage temperature <0°C.     

Experimental study of postmortem change under field conditions: effects of freezing, thawing, and mechanical injury

Understanding the processes of postmortem change in biologic systems is important to the forensic sciences. Previous experimental studies of postmortem change in animals under field conditions made use of animal carcasses that had been incidentally exposed to the effects of freezing and thawing or mechanical damage, or both, and were limited to gross observations. The current study was designed to document intrinsic processes of postmortem change, and the effects of freezing-thawing and mechanical injury, under controlled conditions in the field, using histologic and microbiologic techniques, as well as gross observation. Insect and microbiologic succession sequences, and patterns of decomposition and disarticulation, were observable over time. Previously frozen-thawed animals showed predominantly decay (aerobic decomposition) in the field, while freshly killed animals showed predominantly putrefaction (anaerobic decomposition). Previously frozen animals showed the same sequence, but accelerated rates, of disarticulation. Mechanically injured tissues showed accelerated rates of decomposition. These findings have implications for the interpretation of results of previous studies, as well as the interpretation of human and animal remains subjected to freezing and thawing.     

Determining Postmortem Interval Using Glycoproteinous Adhesion Deposits by Balanus improvisus on Human Skeletal and Dental Remains

An anthropological analysis was conducted on skeletal and dental remains brought to the Galveston County Medical Examiner's office. The skeletal remains were dry, fragmented, and absent of typical fluvial characteristics. During microscopic examination, semitransparent, circular objects were discovered on the dentition, the mandible, tibial plateau, and distal femur. The objects were glycoproteinous adhesions deposited by the acorn barnacle, Balanus improvisus. B. improvisus is an intertidal barnacle found in estuaries in Galveston Bay. Basal diameter of the adhesions on the dentition were significantly smaller than those found on the postcranial bones (p = 0.010), indicating two consecutive cohorts adhered to the bone and dentition. As settlement typically occurs once a year, this would indicate that the remains were in the fluvial environment for at least 375–410 days. It is important in geographic areas that have prevalent fluvial environments that human remains, particularly dentition, are microscopically examined for marine life evidence.     

The Effects of Soil Environment on Postmortem Interval: A Macroscopic Analysis

Abstract:  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.     

Variation in Developmental Time for Geographically Distinct Populations of the Common Green Bottle Fly,Lucilia sericata (Meigen)*

Abstract: Time between death and discovery of remains, or postmortem interval (PMI), can be assessed using blow fly maggot age. Forensic entomologists rely on published, often nonlocal, species‐specific developmental tables to determine maggot age. In a series of common garden experiments, we investigated the developmental rate variation between populations of Lucilia sericata collected from Sacramento, CA, San Diego, CA, and Easton, MA at 16°C, 26°C, and 36°C. For the 16°C trial the time measurement started at egg hatch, while for the higher temperatures the experiment began at oviposition; the wandering stage signified the endpoint for all experiments. The distribution of developmental times differed significantly (ANOVA, p < 0.001) between the three populations within each temperature treatment. We discovered that regional variation of developmental times within a blow fly species exists. This study demonstrates the importance of assembling local population‐specific developmental tables when estimating larval age to determine PMI.