Histological Determination of the Human Origin of Bone Fragments

Abstract:  A frequently encountered task in the forensic scenario is verification of the human origin of severely degraded fragments of bone. In these cases histological methods which consider osteon size and morphology can prove to be useful. The authors in the present study verify the applicability of published algorithms to flat and subadult bones from human, dog, cat, cow, rabbit, sheep, pig, chicken, quail, and turkey samples. Metric analysis was performed on 2031 Haversian canals. Analyses carried out on human samples confirmed a success rate of around 70% on long adult bones; however the percentage of wrong answers was particularly high in the case of newborns and older subadults as well as on flat bones in general. Results therefore suggest that such regression equations should be limited only to bone fragments from long adult bones.     

Radiographic human identification using bones of the hand: a validation study

The 1993 Supreme Court case Daubert v. Merrell-Dow Pharmaceuticals, Inc. underscores the importance of validating forensic science techniques. This research examines the validity of using posterior-anterior radiographs of the hand to make positive identifications of unknown human remains. Furthermore, this study was constructed to satisfy the requirements of Daubert's guidelines of scientific validity by establishing a standard methodology for hand radiograph analysis, testing the technique, and noting rates of error. This validation study required twelve participant examiners from the forensic science community, working independently, to attempt to match 10 simulated postmortem radiographs of skeletonized hands to 40 simulated antemortem radiographs of fleshed cadaver hands. The overall accuracy rate of the twelve examiners was 95%, while their collective sensitivity and specificity were 95% and 92%, respectively. However, the accuracy of each examiner was related to the amount of radiological training and experience of the observer. Six Ph.D. forensic anthropologists and four experienced forensic anthropology graduate students correctly identified all the matches. Participant examiners noted bone morphology, trabecular patterns of the proximal and middle phalanges, and distinctive radiopaque and radiolucent features as the anatomical features that aided the identification process. The hand can be an important skeletal element for radiographic positive identification because it contains 27 individual bones for comparative analysis.     

Determining the human origin of fragments of burnt bone: a comparative study of histological, immunological and DNA techniques.

In situations where badly burnt fragments of bone are found, identification of their human or non-human origin may be impossible by gross morphology alone and other techniques have to be employed. In order to determine whether histological methods were redundant and should be superseded by biomolecular analyses, small fragments of artificially burnt bone (human and non-human) were examined by quantitative and standard light microscopy, and the findings compared with newer biomolecular analyses based on identifying specific human albumin by ELISA and amplifying human mitochondrial DNA by PCR. For quantitative microscopy, reference data were first created using burnt bones from 15 human and 20 common domestic and farm animals. Measured osteon and Haversian canal parameters were analysed using multivariate statistical methods. Highly significant differences were found between values for human and non-human bone, and a canonical discriminant function equation was derived, giving a predicted correct classification of 79%. For the main study, samples of cortical bone were taken from three fresh cadavers, six human skeletons and ten freshly slaughtered animals and burnt by exposure to temperatures ranging from 800 to 1200 degrees C; charred fragments of human cortical bone from two forensic cases were also tested. Quantitative microscopy and canonical discriminant function gave the correct origin of every sample. Standard microscopy falsely assigned burnt bone from one human skeleton and one forensic case to a non-human source, but otherwise gave correct results. Human albumin was identified in five individuals, including one of the forensic cases, but mitochondrial DNA could not be amplified from any of the human bone. No false positive test results were seen with either biomolecular method; and human albumin and mitochondrial DNA were correctly identified in all unburnt control specimens. It was concluded that histological methods were not redundant and that quantitative microscopy provided an accurate and consistent means of determining the human or non-human origin of burnt bone and was more reliable than standard microscopy or the newer immunological and DNA techniques tested here.     

Identification of animal species by protein radioimmunoassay of bone fragments and bloodstained stone tools

In forensics and archaeology, it is important to distinguish human from animal remains and to identify animal species from fragmentary bones and bloodstains. We report blind tests in which a protein radioimmunoassay (pRIA) was used to identify the species of six bone fragments lacking morphological specificity and 43 bloodstained lithic tools, knapped experimentally and soaked in blood of known animal and human origin. The submitters of the bone fragments and the bloodstained tools each listed a number of possible species, from which the testers selected the best match with the pRIA results. All six bone fragments were correctly identified: three humans, a deer, a dog, and a cow. Forty-three tools were stained with blood from a wide variety of species including ungulates, carnivores, a fish, and a bird. On 40 of these 43, at least one species (or blood-free control) was identified correctly. Some of the tools were stained with blood of two different species. A mixture of sheep and musk ox blood was correctly identified; in several other mixtures, only a single species was detected. Two tools with human blood and one with human sweat were correctly reported as human. There was a single false positive (one of three controls reported as weakly bovine) and no false negatives. We conclude that the pRIA technique shows a high degree of accuracy in discriminating human from animal bone fragments and bloodstains and in identifying animal species.     

Discrimination between human and animal DNA: application of a duplex polymerase chain reaction to forensic identification

Identification of a report's species is one of the basic analyses in forensic laboratories. The authors report the case of 6 bone fragments recovered in a wooded area, which were not attributable to 1 animal species on the basis of morphologic examination. The aim of this study was to develop a duplex polymerase chain reaction (PCR) to discriminate human and animal origin of bone fragments. The method is based on the PCR amplification of cytochrome b and a 16S ribosomal mitochondrial DNA fragment, which has never been tested up to now. Our protocol combines a single-round PCR with direct visualization of amplicons in agarose gel, without sequencing analysis of the PCR products. The presence of a single band (359 bp) indicates a nonhuman origin of the sample, whereas 2 bands (157 and 359 bp) indicate a human biologic sample.This method revealed to be useful for forensic purposes because the 16S ribosomal mitochondrial DNA is a small human-specific fragment that is easily amplifiable even with degraded DNA from biologic materials such as old bones.     

Use of solid-phase double-antibody radioimmunoassay to identify species from small skeletal fragments

Protein radioimmunoassay (pRIA) offers the potential to identify species in small skeletal fragments submitted as forensic evidence. The technique consists of protein extraction followed by a solid-phase double-antibody radioimmunoassay using controls of antisera (raised in rabbits) and radioactive (iodine-125) antibody of rabbit gamma globulin (produced in donkeys). Species determination results from evaluation of radioactivity uptake. To demonstrate the potential of this technique, six known bone samples (three human and three nonhuman, including one from a deer [Odocoileus virginianus]) were submitted for blind analysis. pRIA correctly distinguished the human from the nonhuman samples. Using 200 mg or less of each sample, species of the deer specimen was identified correctly, given the choices of cow, deer, dog, goat, and pig.     

Interpretation of ante-mortem stature estimates in South Africans

We developed a simple method for animal species identification of humans, dogs and cats, using a multiplex single-base primer extension reaction in the cytochrome b gene. Using this method, three points of a single nucleotide in the cytochrome b gene were examined in these species using primers of different lengths. Our method was found to be able to successfully identify humans (26 samples), dogs (21 samples) and cats (9 samples), and no differences were found among the samples from each animal species in this study. The amount of template DNA required was over 0.01 ng for humans and dogs, and over 0.1 ng for cats. The present method was able to identify animal species from hair shaft (2 cm) and forensic casework samples (blood stains and hair shafts), and is thus a useful tool for animal species (human, dog and cat) identification in forensic science.     

Adherence of forensic odontologists to the ABFO bite mark guidelines for suspect evidence collection

Boards and associations within forensic science have long been accepted as vehicles for the development and dissemination of protocols and recommendations for practice. Recent controversies surrounding bite mark analyses have brought the methods and practices of forensic dentists to the attention of both the courts and the media. In the mid-eighties the American Board of Forensic Odontology developed guidelines for bite mark analysis in response to unfavorable commentaries on the discipline by legal observers. The purpose of this study is to examine the adherence of board certified and noncertified forensic dentists to the guidelines for collection of evidence from bite mark suspects. A questionnaire was employed during an American Academy of Forensic Sciences meeting. Results showed that, in general, when the odontologists collected evidence they did adhere to the guidelines, although collection of salivary samples was not common. Of concern is the large number of odontologists who do not collect their own evidence from suspects. Police officers or other individuals often perform this task and therefore the guidelines must be disseminated to these groups to ensure that the maximum yield is obtained from bite mark evidence. A review of the materials used to collect evidence is also included with details of applications in forensic science.     

Determination of some somatic body dimensions by osteometric foot measurements

Methods of diagnosis of somatic life-time peculiarities of man (body height, shoulders breadth and dimensions of extremities and their segments) were elaborated on the basis of a study of interrelations between the human body-built features, on the one hand, and measurement characteristics of the foot, on the other hand, by using the modern programs of multi-measurement statistics. Isolated skeleton bones of osseous complexes and fragments of native and burnt bones were the research objects. Footwear and cloths items, which belonged to an actual missing person, can be identification objects. The paper is primarily addressed to forensic medical experts and can be useful for criminalists, anthropologists and archeologists.     

Differentiating fragmented human and nonhuman long bone using osteon circularity

Distinguishing between human and nonhuman bone is important in forensic anthropology and archeology when remains are fragmentary and DNA cannot be obtained. Histological examination of bone is affordable and practical in such situations. This study suggests using osteon circularity to distinguish human bone fragments and hypothesizes that osteons will more closely resemble a perfect circle in nonhumans than in humans. Standard histological methods were used, and circularity was determined using an image analysis program, where circularity was controlled for by Haversian canal measurements. Homogeneity was first tested for multiple variables within human and nonhuman samples. No significant differences were found between human sexes (p = 0.657) or among nonhuman species (p = 0.553). Significant differences were found among intraskeletal elements of both humans (p = 0.016) and nonhumans (p = 0.013) and between pooled samples of humans and nonhumans (p < 0.001). Results of this study indicate that osteon circularity can be used to distinguish between fragmented human and nonhuman long bone.     

Radiological, forensic, and anthropological studies of a concrete block containing bones

Multidisciplinary forensic, anthropological, and radiological studies of bone fragments encased in a concrete block were carried out to determine whether or not the bones were human. Multislice computed tomography (MSCT) investigation was performed before the bones were removed from the concrete. MSCT study pinpointed the location of the bone fragments within the concrete block, which was helpful for their extraction and recovery, and identified most of their types and nature. Osteological study on dry bones provided more accurate identification of the bones and of their side. According to both methods, the human skeletal remains were compatible with those of a child, aged 8-13 years old, with a minimum height of 128 cm. Neither investigation identified sex or racial phenotype. Both studies identified the skeletal remains as consisting of two animal and five human bones. Furthermore, both methods revealed that the concrete completely encased bones, suggesting a secondary burial.     

The use of crossover immunoelectrophoresis to detect human blood protein in soil from an ambush scene in Kosovo

This study examines the survivability of human blood proteins in soils from a year and a half old ambush scene in Kosovo. A total of 72 soil samples were collected, a number of which were directly associated with bone fragments or bullet projectiles. The samples were examined using crossover immunoelectrophoresis (CIEP) to determine the presence of blood protein and species affiliation. Human blood proteins were identified in 44 of the 72 samples (61%) with the majority of the positive observations (29 of 44) found 0.0-4.5 cm below ground surface (65%). Chi-squared and two-sample difference of proportions tests confirmed significant differences between samples with and without associated physical evidence and the presence and depth of human blood proteins. While DNA has largely replaced immunological analysis in forensic analyses, our results suggest that in particular situations, CIEP may still be a valuable tool in criminology.     

A microscopic comparison of fresh and burned bone.

Examination of the microstructure of human bone is useful in estimating age at death in forensic science cases. This technique has been tested and is well accepted, however samples of burned bone may complicate analysis because of possible changes in the microstructure occurring during the burning process. In a comparison of fresh and burned ground thin sections taken from midshaft femorae of eight dissecting-room cadavers of known age and sex, this study finds significant shrinkage of microstructural elements through the burning process. These results are compared to previous work on the subject, which found the microstructural elements to increase through the burning process.     

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。     

Personal identification based on radiographic vertebral features

Personal identification of human remains constitutes about 10% of the normal caseload of any forensic medicine practice. Identification can be achieved by a variety of methods, one of which is the comparison of antemortem and postmortem radiographs. There are numerous accounts of cranial and dental radiographic features useful for identification, whereas the availability of postcranial radiographs and especially plates that depict the vertebral column is less widespread among the forensic community. The authors here review the various vertebral features instrumental in positive identification that can be identified on radiographs of the spine.     

Determination of sex from the clavicle and scapula in a Guatemalan contemporary rural indigenous population

The clavicle has been described as a useful bone for the metric determination of sex of human skeletal remains in a contemporary, predominantly white, North American forensic sample. In this article, measurements of clavicle and scapula are provided for a contemporary Guatemalan rural indigenous sample of forensic origin. Maximum length and circumference at midshaft of the clavicle, and height and width of the glenoid fossa of the scapula, were measured in 35 female and 62 male clavicles, and in 38 female and 65 male scapulae. Discriminant function analysis was used to study sexual dimorphism in this population with a classification purpose. Leave-one-out method (jackknife) matrices produced classification success rates ranging from 85.6% to 94.8%. A comparison with the North American forensic sample showed low percentages of correctly sexed Guatemalan male clavicles, ranging from 29.4% to 54.9%. The choice of an appropriate standard for the metric determination of sex is a crucial step in forensic anthropology.     

Report of the blind trial of the Cetus Amplitype HLA DQ alpha forensic deoxyribonucleic acid (DNA) amplification and typing kit

The AmpliType HLA DQ alpha forensic DNA amplification and typing kit is designed for the qualitative analysis of the human leukocyte antigen (HLA) DQ alpha alleles present in deoxyribonucleic acid (DNA) extracted from forensic samples. The AmpliType kit is the first forensic DNA typing product based on the GeneAmp polymerase chain reaction (PCR) process. The kit was evaluated by five forensic science laboratories (test sites) to assess their ability to perform DNA typing using PCR on sample types typically encountered by forensic laboratories. None of the DNA-containing samples was mistyped. Of the 180 DNA-containing samples analyzed, results were reported for 178 (98.9%). Of the 178 samples with results, all were correctly typed. Two sites did not report a result for one sample each. Four of the five laboratories experienced no significant levels of contamination in the DNA-containing samples. At the one site with the highest number of DNA-containing samples with contamination, the typing results were not compromised. This site was able to correct the contamination problem through simple procedural changes and stricter attention to sterile technique. Blank controls were important to monitor contamination. In conclusion, the trial demonstrated that forensic science laboratories are capable of setting up a PCR-based DNA typing laboratory and successfully using the AmpliType HLA DQ alpha forensic DNA amplification and typing kit to analyze forensic samples.     

人与动物骨组织特征的比较研究

目的研究区别骨骼残片是否为人类骨骼的方法。方法提取人体骨骼及鱼类、两栖类、爬行类、鸟类、哺乳类、灵长类等动物的骨骼标本制做骨骼组织学片,在显微镜下观察,将显微镜下的图像录入电脑进行分析。结果内、外环骨板,骨单位,骨细胞的显微镜下的结构,人类与动物存在明显差别。结论根据骨骼的组织学特征可以区别人类与动物的骨骼。     

The reliability of digitized radiographs for dental identification: a Web-based study

In the era of Daubert and other judicial rulings pertaining to the acceptability of forensic evidence, it is increasingly important that experts are able to testify that their methods have been scientifically tested and that error rates and other factors relating to reliability have been published. The purpose of this study was to determine the reliability of digitized radiographic comparisons for the purposes of dental identification. Participants with various forensic backgrounds and experience levels were passively recruited to the website. Ten forensic identification cases composed of antemortem and postmortem dental radiographs were supplied to examiners using a bespoke website. Participants responded to the cases on two occasions after a one-month washout interval using the ABFO conclusion levels for forensic identifications. A total of 115 first attempts and 87 matched second attempts were received. Of the total responses, 72% were dentally trained respondents who had completed at least one forensic identification case; of these, 38% were experienced forensic dentists who had completed more than 25 identifications. Data relating to accuracy, intra- and inter-examiner agreement, and the effect of case difficulty are presented. Mean accuracy was 85.5% for all cases, with the experienced forensic dentists obtaining a 91% success rate. The inter-examiner agreement on the negative identification cases was classified as poor. The data suggest that dental identifications resulting from the comparison of postmortem and antemortem radiographs are valid, accurate, and reliable when undertaken by experienced odontologists.     

Multiplex-direct PCR assay for foodborne pathogen identification: An application in forensic investigation

Foodborne pathogens present serious concerns to human health and can even lead to fatalities. Microbial forensic science thus plays an important role in consumer protection, food security, and even in litigation. The gold standard for pathogen identification – bacterial culture – is costly and time-consuming. A cheaper and quicker alternative will benefit both forensic science and medical diagnosis. In this study, we developed and validated a molecular-based method termed ‘multiplex-direct PCR assay’ to simultaneously detect three common foodborne pathogens – Escherichia coli O157:H7, Campylobacter jejuni, and Listeria monocytogenes. Three previously reported species-specific primer pairs were modified and used to directly amplify samples without DNA extraction. The assay was also validated for its specificity, sensitivity, and applied to test several samples obtained from a local market and clinical samples. The results showed the expected PCR fragments of approximately 490, 343, and 209 bp for E. coli O157:H7, C. jejuni, and L. monocytogenes, respectively. The assay was specific to the targeted pathogens and was sufficiently sensitive and robust to effectively analyze market samples. The whole process took less than 1 h to complete indicating that the assay is suitable for reliable, rapid, and inexpensive identification of these three foodborne pathogens, which could be useful in microbial forensic investigation.