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.     

法医骨组织学研究

在实际检案中 ,当现场发现的骨骼残片体积较小时 ,用解剖学观察无法进行骨骼残片的法医鉴定 ,需使用骨组织学的方法进行骨骼残片的个体识别。目前 ,这是法医人类学中一门较活跃的领域 ,即法医骨组织学。法医骨组织学的内容主要包括两个方面 :(1)骨骼残片是否属于人类骨骼 ,或属于何种动物骨骼。这方面的研究包括人类骨骼的组织学特征研究及不同动物的组织学特征研究。 (2 )人类骨骼个体识别的组织学研究。这方面的研究主要包括 ,人类骨骼的组织学特征的年龄判断 ,例如股骨、胫骨、肱骨、锁骨等 ,以及使用骨组织学方法 ,进行人类骨骼的年龄评价的准确性研究。本文对上述内容进行了综述。     

A Comparison of Histomorphometric Data Collection Methods*

Abstract: Although many variables that skeletal biologists examine have been standardized, the actual techniques used to collect these data from bone thin sections vary. This project compares different methods of obtaining data (relative cortical area values) for histomorphometric research. One visual and three digital methods of histomorphometric data collection are compared: (i) Merz microscopic eyepiece counting reticule, (ii) flatbed scanner, (iii) overlaying multiple images of a thin section, and (iv) digital SLR camera with macro settings. The discussion includes a comparison of usability factors such as cost, time, user‐experience, and ease‐of‐use, which vary for each method. Values from the different methods are compared using ANOVA tests to evaluate inter‐method, inter‐observer, and intra‐observer variability. Intra‐observer error was greater for the microscopic method, although the error values are concomitant with experience. We found no statistically significant differences between the four methods examined, but certain caveats must be addressed when these methods are used.     

Differences among species in compact bone tissue microstructure of mammalian skeleton: use of a discriminant function analysis for species identification

In order to develop an identification key for distinguishing between several mammalian species, bone structure of their compact bone tissue was analyzed using qualitative and quantitative characteristics. Ninety femora of adult male humans, pigs, cows, sheep, rabbits, and rats were studied. The average area, perimeter, minimum, and maximum diameter of 1863 Haversian canals and 1863 secondary osteons were measured using a digital image device. The observed data were first used to evaluate inter- and intraspecies diversity. After that, we applied a discriminant function analysis for differentiation of the species by these variables. Classification functions for investigated species give cross-validated correct classification rates for 76.17% of cases. This percentage value can be increased by integrating conclusions from the qualitative analysis.     

Quantification of Osteon Morphology Using Geometric Histomorphometrics

Many histological methods in forensic anthropology utilize combinations of traditional histomorphometric parameters which may not accurately describe the morphology of microstructural features. Here, we report the novel application of a geometric morphometric method suitable when considering structures without anatomically homologous landmarks for the quantification of complete secondary osteon size and morphology. The method is tested for its suitability in the measurement of intact secondary osteons using osteons digitized from transverse femoral diaphyseal sections prepared from two human individuals. The results of methodological testing demonstrate the efficacy of the technique when applied to intact secondary osteons. In providing accurate characterization of micromorphology within the robust mathematical framework of geometric morphometrics, this method may surpass traditional histomorphometric variables currently employed in forensic research and practice. A preliminary study of the intersectional histomorphometric variation within the femoral diaphysis is made using this geometric histomorphometric method to demonstrate its potential.     

中国汉族人耻骨性别判定的研究

目的为了探讨耻骨性别判定的更为简便可靠的方法。方法对96副已知生前确切性别的耻骨进行了性别差异的研究,其中男性耻骨73副,女性耻骨23副。测量项目有:缘枝角,耻骨宽,耻骨联合面的高及厚。结果将耻骨的测量数据输入电脑,用SPSS软件,对耻骨的测量数据进行了统计分析,建立了耻骨性别判定的判别方程。结论其性别判别率最高可达95.5%,该方法可以用于法医学实践。     

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.     

烧骨组织形态变化及DNA技术在个体识别中的应用

烧骨在火灾、焚尸、交通、爆炸等案件和事故的检材中具有特殊的地位。通过对不同条件焚烧下烧骨组织形态及DNA变化规律的研究,可为法医实践中烧骨的种属鉴定、性别及年龄判定提供准确的依据和标准,同时可利用残存的基因位点对烧骨残块进行个体识别和同一认定。烧骨DNA的提取方法及检测技术也在不断探索和改进。本文对烧骨在形态学、组织学和分子生物学水平研究进展以及烧骨评测的方法、技术进行概述,旨在为法医实践及进一步研究提供新的方法和思路。     

Inter-species variation in bone mineral behavior upon heating

The characterization of inter-species variation in bone mineral (b-HAP) is of relevance to forensic science and archaeology, but has not previously been widely explored. Results of an investigation into unheated bone mineral and behavior of bone upon heating for 12 animal species (including human) demonstrate that b-HAP characteristics, quantitatively measured using X-ray diffraction (XRD) analysis, exhibit significant inter-species variation. Human bone was found to be significantly different to all other species in terms of b-HAP lattice parameter values from unheated and heated bone and in terms of recrystallization behavior of b-HAP upon heating bone to 600°C. The amounts of b-HAP thermal decomposition products were also significantly different for human bone heated to 1400°C compared to those obtained for most other species. Therefore, there is potential for the development of an XRD-based method of species identification, particularly one that distinguishes human from non-human bone.     

Assessment of Maceration Techniques Used to Remove Soft Tissue from Bone in Cut Mark Analysis

Maceration techniques employed in forensics must be effective without compromising the bone's integrity and morphology, and prevent destruction of evidence. Techniques must also be fast, safe, easily obtainable and inexpensive; not all techniques currently employed are appropriate for forensic use. To evaluate the most suitable approach, seven techniques including current and new methodologies were applied to fresh, fleshed porcine ribs exhibiting cut marks. A sample size of 30 specimens per technique was examined under scanning electron microscopy at the cut mark and the surrounding uncompromised regions; a scoring system of effectiveness was applied. The previously unpublished microwave method fared best for bone and cut mark preservation. Sodium hypochlorite destroyed cut marks, and was deemed unsuitable for forensic analysis. No single technique fulfilled all criteria; however, this study provides a benchmark for forensic anthropologists to select the most appropriate method for their situation, while maintaining the high standards required by forensic science.     

A Case of Contested Cremains Analyzed Through Metric and Chemical Comparison

Since the 1980s, cremation has become the fastest growing area of the U.S. funeral industry. At the same time, the number of litigations against funeral homes and cremation facilities has increased. Forensic anthropologists are often asked to determine whether the contents of an urn are actually cremated bone, and to address questions regarding the identity of the remains. This study uses both metric and chemical analyses for resolving a case of contested cremains. A cremains weight of 2021.8 g was predicted based on the decedent's reported stature and weight. However, the urn contents weighed 4173.5 g. The urn contents also contained material inconsistent with cremains (e.g., moist sediment, stones, ferrous metal). Analysis using XRD and SEM demonstrated that the urn contained thermally altered bone as well as inorganic material consistent with glass fiber cement. Although forensically challenging, cremains cases such as this one can be resolved using a multidisciplinary approach.     

Testing Discriminant Functions for Sex Determination from Deciduous Teeth*,†

Abstract: Three studies have proposed discriminant functions for sex determination from deciduous tooth crown dimensions, and this study tests the existing functions on a sample of 46 Portuguese immature skeletons of known sex, aged from birth to 10 years. Deciduous teeth were measured in their mesiodistal and faciolingual crown dimensions, and percentage of correct allocation accuracy in determining sex using each specific function was determined. Discriminant functions were also calculated from data collected for this study and tested using cross‐validation. Results show poor overall accuracy (33.3–75%) and poor cross‐validation (46.2–60.0%). This is related to low sexual dimorphism in deciduous tooth crown size, as well as differences in degree of sexual dimorphism and in overall tooth size between different samples. For these reasons, deciduous crown size does not seem to show significant forensic value as discriminator of sex, particularly when methods developed on one population are applied to individuals of another population.     

Assessment of intra- and intercostal variation in rib histomorphometry: its impact on evidentiary examination

Rib histological age estimation requires the evaluation of the middle third of the sixth rib. Human ribs have thin cortices and, when recovered, are often fragmented or absent, making it difficult to identify a specific midthoracic rib. This research explores the amount of microstructure variation in the middle third of the midthoracic ribs and determines whether the sixth rib age prediction equation can be applied to non-sixth ribs with similar accuracy. The amount of variability must be evaluated in order to meet the criterion for evidentiary examination. The sample consists of 120 cortical bone cross-sections from the middle third of ribs 3-8 removed from 20 cadavers. For each rib, osteon population densities (OPDs) and associated age estimates were calculated. The results demonstrate that non-sixth ribs can provide similar OPD values compared with those of the sixth ribs; however, individual variation proved to be significantly associated with bias, suggesting that individual factors influence the magnitude and direction of bias in non-sixth rib OPD values. This demonstrates the importance of evaluating multiple cross-sections (both intra- and inter-rib) to estimate age due to the normal remodeling variation within individuals.     

Microscopic Residues of Bone from Dissolving Human Remains in Acids

Dissolving bodies is a current method of disposing of human remains and has been practiced throughout the years. During the last decade in the Netherlands, two cases have emerged in which human remains were treated with acid. In the first case, the remains of a cremated body were treated with hydrofluoric acid. In the second case, two complete bodies were dissolved in a mixture of hydrochloric and sulfuric acid. In both cases, a great variety of evidence was collected at the scene of crime, part of which was embedded in resin, polished, and investigated using SEM/EDX. Apart from macroscopic findings like residual bone and artificial teeth, in both cases, distinct microscopic residues of bone were found as follows: (partly) digested bone, thin‐walled structures, and recrystallized calcium phosphate. Although some may believe it is possible to dissolve a body in acid completely, at least some of these microscopic residues will always be found.     

Histological age prediction from the femur in a contemporary Dutch sample. The decrease of nonremodeled bone in the anterior cortex

This paper presents an uncomplicated and minimally invasive method for age-at-death determination in a contemporary Dutch (West European) population, by modifying the approach of assessment based on the age-related remodeling of bone tissue. In contrast to the usual "osteon count," a "non-remodeled tissue count" is undertaken. To optimize the method, proper zeroing of the polarization filter set of the microscope is essential. Instructions for setting the filters are given. A sample of femoral shaft segments totaling 162 individuals with ages ranging from 15 to 96 years is analyzed. Subperiosteal quantitative assessments are recorded at the most anterior point of the femoral shaft and also at points 25 degrees to the left and to the right of that point. Interobserver agreement in the assessments shows an acceptable degree of correlation. Bone remodeling with age does not progress in a linear, but in a curvilinear manner. Dependence of predicted age on nonremodeled surface counts in the analyzed areas of the anterior cortex of the femur appears to be significant. A set of regression equations is given. Sex can be ignored in age prediction. The small but statistically significant dependence of predicted age on cadaver length corresponds with the present strong secular increase in stature in the Netherlands. A concise catalogue with micrograph examples for every 10-year period in life is available upon request.     

Morphometrics of the hyoid bone for human sex determination from digital photographs

The identification of unknown remains is very important. When unknown remains are found, anthropologists first determine their sex and age. The sex of most skeletons is determined by their shape. In the hyoid bone, the shape is sex related, so it can be used forensically to determine the sex. This study focused on sex-based morphometry of the hyoid bone in Koreans using digital photographs. Hyoid bones from 52 males and 33 females were examined. For each subject, we took 34 measurements from photographs using a computer program, and the data were analyzed statistically using SPSS 11.0. Twenty-one of 34 measurements had significant sex differences (p<0.05). The discriminant functions based on three measurements (X(1)-X(3)) were as follows: The accuracy of discriminant functions is 88.2% in both groups, so these can be used to distinguish males from females in a statistically significant manner.     

Assessment of Wear‐Related Features of the Kerf Wall from Saw Marks in Bone*†

Abstract: Analyses of saw marks in bone may yield important information about the class characteristics of saws used in postmortem dismemberments, yet little research has been directed at identifying saws’ individualizing characteristics. This study adds to existing saw mark analysis methodologies by examining wear‐related features of kerf walls using light‐ and environmental scanning electron microscopy. A crosscut saw and hacksaw were used to create sequences of 30 cuts in bone; these sequences reveal patterns of progressive loss of fine details of kerf wall morphology with increasing saw blade wear, because of the rounding of sharp points and edges. Nevertheless, diagnostic kerf wall features used to establish class characteristics persist despite these wear‐related changes. Unsuccessful attempts at statistical analysis of wear‐related changes, based on striae width and density, suggest these patterns are not readily quantifiable. Additionally, despite the scanning electron microscope’s superior imaging capabilities, it provided few practical, methodological gains over traditional light microscopy.     

Identification from Chest X‐Rays: Reliability of Bone Density Patterns of the Humerus*

Abstract: A critical review of Kahana and Hiss’ study on identification from bone trabecular pattern and a test of their method conducted on the humerus are presented. Bone trabecular pattern was studied through the generation of a numerical file representing the gray scale. Using the correlation coefficient, several pairwise comparisons between numerical files were performed. The test gave nearly 30% of incorrect exclusions (the method did not recognize couples of radiographs belonging to the same subject) and 50% of misidentifications (the method recognized couples of radiographs belonging to different subjects, as belonging to the same subject); therefore, this research shows that at the present time, it is not possible to safely quantify identification through bone density patterns, of the proximal humerus taken from thoracic X‐rays. Thus, an “easy”—but dangerous—use of trabecular density patterns on this specific type of radiogram as an identification method should be currently avoided.     

The use of material culture to establish the ethnic identity of victims in genocide investigations: a validation study from the American Southwest*

Successful prosecution of genocide requires that the victims constitute one of four protected groups: national, religious, ethnic, or racial. Establishing victim identity in prior trials has relied on positive identification of decedents, been largely presumptive, or was based on untested methodology. This report details a validation study of one untested method: the use of material culture in establishing ethnic identity. Classes of clothing and personal effects were scored on 3,430 individuals of known Hispanic or White ancestry from autopsy records in New Mexico. Significant differences were seen in evidence of language, nationality, and religious affiliation between the two groups, as well as clothing types and currency. Predictive models used to estimate ethnic identity in random, blind subsets produced an overall accuracy of 81.5% and estimates of 61-98% in specific subsets. Results suggest material culture, when present, can provide reliable evidence of ethnic affinity in genocide investigations.     

The effects of corrosive substances on human bone, teeth, hair, nails, and soft tissue

Abstract: This research investigates the effects of household chemicals on human tissues. Five different human tissues (bone, tooth, hair, fingernails, and skin/muscle/fat) were immersed into six different corrosive agents. These agents consisted of hydrochloric acid, sulfuric acid, lye, bleach, organic septic cleaner, and Coca‐Cola^® soda. Tap water was used as a control. Tissue samples were cut to consistent sizes and submerged in the corrosive liquids. Over time, the appearance, consistency, and weight were documented. Hydrochloric acid was the most destructive agent in this study, consuming most tissues within 24 h. Sulfuric acid was the second most destructive agent in this study. Bleach, lye, and cola had no structural effects on the hard tissues of the body, but did alter the appearance or integrity of the hair, nails, or flesh in some way. The organic septic cleaner and tap water had no effect on any of the human tissue tested during the timeframe of the study.