Forensic scatology: preliminary experimental study of the preparation and potential for identification of captive carnivore scat

Carnivore scats recovered from animal attack and/or scavenging contexts frequently contain forensic evidence such as human bone fragments. Forensic cases with carnivore involvement are increasingly prevalent, necessitating a methodology for the recovery and analysis of scat evidence. This study proposes a method for the safe preparation of carnivore scat, recovery of bone inclusions, and quantification and comparison of scat variables. Fourteen scats (lion, jaguar, lynx, wolf, and coyote) were prepared with sodium-acetate-formalin fixative; analytical variables included carnivore individual, species, body size, and taxonomic family. Scat variables, particularly bone fragment inclusions, were found to vary among carnivore individuals, families, species, and sizes. The methods in this study facilitate safe scat processing, the complete recovery of digested evidence, and the preliminary identification of involved animals. This research demonstrates that scat collected from forensic contexts can yield valuable information concerning both the victim and the carnivore involved.     

Mitochondrial DNA amplification success rate as a function of hair morphology

This study examines the amplification success rate of mitochondrial DNA from human head hair with respect to their potential for forensic application. Mitochondrial DNA was isolated using a Chelex-based extraction method and amplified using the LINEAR ARRAY duplex PCR system. The particular focus of this study was to characterize the morphological features of human head hair in order to further the understanding of the factors that influence amplification success rate in hair tissue using the LINEAR ARRAY duplex PCR system. 2554 head hairs from 132 individuals representing four population groups were amplified. The hair samples were characterized as follows: 1251 were identified microscopically as telogen hairs and 1303 were classified as hairs without roots (removed before extraction). Amplification success was assessed as a function of several independent variables: morphological characteristics; telogen root versus no root; donor age; scalp origin; use of cosmetic hair treatments; and race of the donor. The results show that a positive correlation exists between amplification success and the presence of a telogen root. Combining the amplification success with either the original or optimized protocol, telogen hairs result in an overall success rate of 77.5% compared with 65% for hairs with no roots. Controlling for telogen hairs, the findings indicate that the overall success rate is independent of cosmetic hair treatments; medulla structure; shaft length, diameter, and volume; and scalp origin. Conversely, the age of the donor, the race of the donor, and hair pigmentation all contribute to a variation in amplification success rate.     

Development of a Protein‐based Human Identification Capability from a Single Hair

Shed human hair (lacking root nuclear DNA) frequently contributes important information to forensic investigations involving human identification. Detection of genetic variation observed in amino acid sequences of hair proteins provides a new suite of identity markers that augment microscopic hair analysis and mitochondrial DNA sequencing. In this study, a new method that completely dissolves single hairs using a combination of heat, ultrasonication, and surfactants was developed. Dissolved proteins were digested and genetically variant peptide (GVP) profiles were obtained for single hairs (25 mm) via high‐resolution nanoflow liquid chromatography‐based mass spectrometry and a novel exome‐driven bioinformatic approach. Overall, 6519 unique peptides were identified and a total of 57 GVPs were confirmed. Random match probabilities ranged between 2.6 × 10^?2 and 6.0 × 10^?9. The new bioinformatic strategy and ability to analyze GVPs in forensically relevant samples sizes demonstrate applicability of this approach to distinguish individuals in forensic contexts.     

Microscopical discrimination of human head hairs sharing a mitochondrial haplogroup

In forensic analyses, determining the level of consensus among examiners for hair comparison conclusions and ancestry identifications is important for assessing the scientific validity of microscopical hair examinations. Here, we present data from an interlaboratory study on the accuracy of microscopical hair comparisons among a subset of experienced hair examiners currently analyzing hair in forensic laboratories across the United States. We examined how well microscopical analysis of hair can reliably be used to differentiate hair samples, many of which were macroscopically similar. Using cut hair samples, many sharing similar macroscopic and microscopic features, collected from individuals who share the same mitochondrial haplogroup as an indication of genetic relatedness, we tested multiple aspects that could impact hair comparisons. This research tested the extent to which morphological features related to ancestry and hair length influence conclusions. Microscopical hair examinations yielded accurate assessments of inclusion/exclusion relative to the reference samples among 85% of the pairwise comparisons. We found shorter hairs had reduced levels of accuracy and hairs from populations examiners were not familiar with may have impacted their ability to resolve features. The reliability of ancestry determinations is not yet clear, but we found indications that the existing categories are only somewhat related to current ethnic and genetic variation. Our results provide support for the continued utility of microscopical comparison of hairs within forensic laboratories and to advocate for a combined analytical approach using both microscopical analysis and mtDNA data on all forensic analyses of hair.     

Low-cost direct PCR for aged and processed wildlife sample analysis

Direct PCR has been used successfully in wildlife forensic DNA analysis from several types of biological samples using specialized, commercial direct PCR kits. This is attributed to the proprietary chemicals provided in the kits such as pre-PCR buffer and modified DNA polymerases. These reagents can be expensive, thereby limiting their widespread adoption in developing countries, where wildlife crimes are often encountered. We report on a study to evaluate the possibility of using low-cost direct PCR assay for degraded and processed wildlife sample analysis. Phire^® and Q5^® polymerases were used, due to their relatively low cost, for direct amplification of six aged and processed sample types (dried skin, 30-year old hair, muscle tissue, bone, trace blood mixed in vodka, and dried soft antler). The result indicated that Phire^® Hot Start II DNA polymerase and Q5^® DNA polymerase performed similarly to commercially available direct PCR kit. The low-cost amplification could efficiently identify species origin from all aged and processed samples. We observed a rate of more than 80% amplification success and high PCR product concentrations sufficient for further sequencing. The assay proved to be cost effective and robust; thus, we expect it to be adopted by wildlife forensic laboratories in developing countries.     

Trichobezoar in Vagina: Assessment for Child Sexual Abuse and Diagnostic Result of Forensic Science

Vaginal discharge and bleeding in children require a through and thoughtful evaluation to diagnose the underlying problem including infections, sexual abuse, and vaginal foreign bodies. We report a 6‐year‐old girl presenting with bloody vaginal discharge, carefully evaluated for sexual abuse, and finally diagnosed as a vaginal foreign body after vaginoscopy. A rolling hair ball was extracted from the vagina and was diagnosed as trichobezoar pathologically without any endo–ecto‐mesodermal residual tissue. The hair ball was genetically detected and diagnosed to belong herself by containing no foreign structure. Child sexual abuse was ruled out by forensic interview at CAC and report of forensic science that reported genetic structure belonging to the child. Medicolegal assessment helped in final diagnosis to exclude child sexual abuse.     

The ultrastructure of tissue attached to telogen hair roots

Most tissues encountered in forensic biology laboratories have been previously characterized with electron microscopy due to their medical importance. Anagen hair root cells, epithelial cells, erythrocytes, neutrophils, osteocytes, and spermatozoa have received considerable research attention at the ultrastructural level. There is no literature indicating that cells attached to removed telogen hair roots have been characterized with transmission electron microscopy. Nonetheless, telogen hairs are a frequent submission item to forensic laboratories for DNA typing. The amount of tissue attached to a telogen hair root usually determines whether that hair is suitable for nuclear DNA typing methods or mitochondrial DNA typing methods. This study used transmission and scanning electron microscopy to characterize the tissues found in three commonly occurring telogen hair root forms. The tissues were found to consist of keratinized remnant follicle, nonnucleated epithelial cells, nucleated epithelial cells, and trichilemmal keratin. These findings were consistent with the known principles of hair follicle regression. The recognition of the root structures that contain these specific tissue types may assist in the DNA typing of telogen hairs inasmuch as the quality of tissue present may be more important than the amounts of tissue present.     

Forensic determination of hair deposition time in crime scenes using electron paramagnetic resonance

Several types of biological samples, including hair strands, are found at crime scenes. Apart from the identification of the value and the contributor of the probative evidence, it is important to prove that the time of shedding of hair belonging to a suspect or victim matches the crime window. To this end, to estimate the ex vivo aging of hair, we evaluated time‐dependent changes in melanin‐derived free radicals in blond, brown, and black hairs by using electron paramagnetic resonance spectroscopy (EPR). Hair strands aged under controlled conditions (humidity 40%, temperature 20–22°C, indirect light, with 12/12 hour of light/darkness cycles) showed a time‐dependent decay of melanin‐derived radicals. The half‐life of eumelanin‐derived radicals in hair under our experimental settings was estimated at 22 ± 2 days whereas that of pheomelanin was about 2 days suggesting better stabilization of unpaired electrons by eumelanin. Taken together, this study provides a reference for future forensic studies on determination of degradation of shed hair in a crime scene by following eumelanin radicals by utilizing the non‐invasive, non‐destructive, and highly specific EPR technique.     

Predicting Phenotype from Genotype: Normal Pigmentation*

Abstract: Genetic information in forensic studies is largely limited to CODIS data and the ability to match samples and assign them to an individual. However, there are circumstances, in which a given DNA sample does not match anyone in the CODIS database, and no other information about the donor is available. In this study, we determined 75 SNPs in 24 genes (previously implicated in human or animal pigmentation studies) for the analysis of single‐ and multi‐locus associations with hair, skin, and eye color in 789 individuals of various ethnic backgrounds. Using multiple linear regression modeling, five SNPs in five genes were found to account for large proportions of pigmentation variation in hair, skin, and eyes in our across‐population analyses. Thus, these models may be of predictive value to determine an individual’s pigmentation type from a forensic sample, independent of ethnic origin.     

Preliminary study of hair form of Japanese head hairs using image analysis

The use of average curvature measurements for the forensic comparison of curly hairs has been reported, but a method, in which various types of hair form are quantitatively examined and objectively interpreted for hair comparison, has not been reported to date. In the present study, numerical data on hair form from Japanese subjects were obtained by image analysis and a morphological comparison of these head hairs was investigated. Head hairs obtained from eight Japanese males were measured for length (L), distance (D) and area (A) using a Kontron Imaging System KS400. From the three measurements mentioned above, three indexes, L/D, A/D and 2(A/L), were examined. The inter-individual variations for each value were investigated by a t-test and the availability of six values for the forensic comparison of hair form was evaluated by a stepwise linear discrimination analysis. Six values obtained from hair form by an image analysis showed large intra-individual variations. However, these six values were found to be useful for discriminating between two individuals, since the six values showed larger inter-individual variations than intra-individual variations. Discrimination on each comparison using a stepwise linear discrimination analysis was performed for some of the values and the results indicated conspicuous inter-individual variations between the two individuals. On 11 of 28 comparisons, 30 hairs from one individual could be completely distinguished from hairs of another individual, when a two-way comparison was employed. These results confirm that hair form could be quite useful in the forensic comparison of hair morphology, and suggest that numerical data obtained from hair form by image analysis are very important values for constructing a screening procedure for evidential hairs. The use of an objective measure of hair form will be especially useful for Japanese head hairs since they are generally thought to show very limited variation in morphological features.     

Characterization of Mitochondrial DNA Sequence Heteroplasmy in Blood Tissue and Hair as a Function of Hair Morphology*,†,‡

Abstract: This study characterizes mitochondrial DNA (mtDNA) sequence heteroplasmy in blood tissue and hair as a function of hair morphology. Bloodstains (127 individuals) and head hairs (128 individuals) were typed using the mtDNA LINEAR ARRAY? assay. A total of 1589 hairs were interpreted: 1478 (93%) were homoplasmic and 111 (7%) exhibited heteroplasmy at one or more positions. Seventy‐one percent (82/116) of individuals were homoplasmic, whereas 29% (34/116) exhibited heteroplasmy in at least one hair. The results demonstrate intra‐ and inter‐tissue differences in heteroplasmy within individuals. Sequence heteroplasmy among hairs from each individual varied from 0 to 90%; the frequency does not differ significantly with population group, cosmetic treatment, age, gender, medulla morphology, region of the scalp, hair growth phase, or, when comparing living and deceased donors. However, the results support a correlation between heteroplasmy and hair pigmentation; typically, lighter‐pigmented hairs exhibit a higher incidence of sequence heteroplasmy compared to darker hairs.     

Forensic Paleontology: A Tool for “Intelligence” and Investigation

Paleontology and facies analysis proved to be useful tools in activities of intelligence and investigation on some criminal cases, as well as in in‐court activities, thus defining the “forensic paleontology” area of study. The definition was given by analyzing its possible specific applications and excluding some marginal activities. The reliability of forensic paleontology was then assessed in light of the results achieved in some actual cases and in an ad hoc simulation. The investigated cases concerned intelligence and ordinary law enforcement activities. Special attention was paid to crimes against the cultural heritage. Ex post re‐examination of the cases substantiated the value of this scientific branch in investigations, while stressing the possible difficulties in explaining its results to lay persons. Therefore, careful preparation of technical and linguistic preliminary notes for judges, prosecutors, and lawyers as well as a special training for consultants are recommended before presenting results as exhibits in in‐court cross‐examinations.     

Peer‐Reviewed Forensic Consultation in Practice: Multidisciplinary Oversight in Common Expertise

The fallibility of forensic science consultation is an ongoing and major justice concern. Prospective peer‐reviewed forensic consultation has over 10 years of application in American criminal and civil courts, adapting from the traditional oversight of teaching hospitals, rules of evidence and discovery, conventions of testimony of expert witnesses, and attorneys' overall trial strategy. In systematizing heightened oversight, this process ensures greater accountability in forensic science consultation. The integration of peer reviewers' complementary expertise and experience enhances the sophistication and overall quality of assessment. Forensic examination frequently involves the interface of different specialties. Multidisciplinary peer review augments expert proficiency with that of professional peers having different vantage points from relevant scientific disciplines. This approach ensures greater sophistication of a case inquiry, built‐in accountability, and streamlined processes when multiple experts are necessitated. Here, the authors present examples of several cases and the primary and secondary benefits of this collaborative, rigorous, cross‐disciplinary exercise.     

A Method for Estimating Sex Using Metric Analysis of the Scapula*

Abstract: The most accurate and precise methods for the assessment of age and stature often require knowledge of sex. Thus, being able to correctly identify sex from skeletal remains is critical in the forensic context. The presence of the os coxae or skull can never be guaranteed, making the development of reliable methods of sex estimation using other skeletal elements necessary. Using a 724 individual calibration sample from the Hamann‐Todd collection, this study identifies sexual dimorphism in the human scapula, and presents a new five‐variable discriminant function for sex estimation. The overall accuracy of this method proved to be 95.7% on the cross‐validated calibration sample, 92.5% on an 80 individual test sample from the Hamann‐Todd collection, and 84.4% on a 32 individual test sample from the skeletal collection of the Wichita State University Biological Anthropology Laboratory. Additionally, a slightly less accurate two‐variable model was developed and has cross‐validated accuracy of 91.3%.     

The Optimized Summed Scored Attributes Method for the Classification of U.S. Blacks and Whites: A Validation Study

Recently, Hefner and Ousley (2014) introduced the optimized summed scored attributes (OSSA) method that maximizes between‐group differences in U.S. black and white populations by dichotomizing six cranial morphoscopic trait scores. This study tests OSSA using an independent skeletal sample (Hamann‐Todd, n = 208) and positively identified forensic cases (Mercyhurst University, n = 28, and New York City Office of Chief Medical Examiner, n = 38). An evaluation of trait frequencies suggests shifting the heuristically selected sectioning point separating U.S. black and white populations from ≤ 3 to ≤ 4. We found a total correct classification of 73.0% (B = 50.9%, W = 89.2%) using the originally suggested sectioning point of ≤3, while the total correct classification increases to 79.2% (B = 80.2%, W = 78.5%) with a modified sectioning point of ≤4. With the increased total correct classification and reduced classification bias between ancestry groups, we suggest the modified sectioning point of ≤4 be used when assessing ancestry in forensic unknowns.     

Morphology of hairs on the head and other parts of the body in the residents of Africa

The morphology of hairs from the head, beard, chest, armpits, and pubis was studied in indigenous population of Sudan, Sierra-Leone, Cote d'Ivoir, Ruanda, Nigeria, Gambia, Kenya, Uganda, Burundi, Zaire, Southern African Republic, Angola, Benin, and Guinea Bissau. A total of 327 hair specimens were examined. A number of new parameters of hairs were determined: thickness, number of cuticle pattern lines per mm, optic edge pattern, cuticle patterns, methods for detecting the cortical and medullar layers, width of hair layers and their transverse sections. The resultant morphological signs are new specific signs for African residents (Negroid) which can be used in forensic medical studies for identifying the race appurtenance of hairs.     

Validation of a multiplex PCR assay for the forensic identification of Indian crocodiles

A dependable and efficient wildlife species identification system is essential for swift dispensation of the justice linking wildlife crimes. Development of molecular techniques is befitting the need of the time. The forensic laboratories often receive highly ill-treated samples for identification purposes, and thus, validation of any novel methodology is necessary for forensic usage. We validate a novel multiplex polymerase chain reaction assay, developed at this laboratory for the forensic identification of three Indian crocodiles, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, following the guidelines of Scientific Working Group on DNA Analysis Methods. The multiplex PCR was tested for its specificity, reproducibility, sensitivity, and stability. This study also includes the samples treated with various chemical substances and exposed to various environmental regimes. The result of this validation study promises this technique to be an efficient identification tool for Indian crocodiles and therefore is recommended for forensic purposes.     

Stature Estimation from Hand Dimensions in a Han Population of Southern China*

Abstract: To analyze the relationship between stature and hand dimensions for forensic applications, the stature and hand dimensions of 400 healthy adults aged between 20 and 25 years were measured in a Han population of Southern China. The mean values of the stature are 170.49 and 159.72 cm in the men and the women, respectively. The statistically significant differences between the right‐ and the left‐hand dimensions were not observed in the men, whereas the bilateral differences are statistically significant in female hand dimensions. The correlation coefficients were found to be statistically significant for the hand dimensions in both the sexes. The hand length showed higher correlation coefficients than the hand breadth in both sexes. Linear and multiple regressions were developed in this study; multiple regressions showed higher correlation coefficients than linear regressions. Two regression models could be used to estimate the stature from the hand dimensions in this population.     

Pacemaker/defibrillator evaluation at Los Angeles County Department of Coroner*

Pacemakers and implantable cardioverter-defibrillators (ICDs) are implanted medical devices for the treatment of cardiac arrhythmias. These devices are now commonly encountered in the postmortem situation. The Los Angeles County Department of the Coroner uses the services of a cardiac electrophysiology consultant to interrogate pacemakers and ICDs, producing a detailed picture of the cardiac events recorded by the device. We have used this method to evaluate 20 cases where the ICD or pacemaker provided significant information. We report four forensic cases to illustrate the different applications of pacemaker interrogation and its contribution in forensic investigation. This technique was used to establish identification of the decedent, to determine the cause and time of death, and to determine whether device malfunction could have played a role in the death. We conclude that detailed evaluation of the pacemaker or ICD in selected cases may provide essential information to the forensic pathologist.