Quantitative evaluation of endometrium-expressed mRNAs for the purpose of discriminating between menstruation and traumatic vaginal injury in sexual assault cases.

In sexual assault cases, it is crucial to discriminate between peripheral blood and menstrual blood to provide evidence for vaginal intercourse with traumatic injury. In this study, the menstrual blood mRNA markers progestagen-associated endometrial protein (PAEP), matrix metallopeptidase 7 (MMP7), and left-right determination factor 2 (LEFTY2) were evaluated by quantitative RT-PCR (RT-qPCR) for the discrimination of menstrual blood from peripheral blood and vaginal fluid. As a result, all markers with cutoff delta cycle quantification (ΔCq) values were specifically determined in menstrual blood among forensically relevant body fluids. Even though the changes in the expression levels of each marker differed during the menstrual cycle, all markers were determined to be positive in most of the randomly collected menstrual blood samples that were analyzed. Additionally, the markers with proposed cutoff ΔCq values could discriminate between menstrual blood and peripheral blood-mixed vaginal fluid samples. The determination of positive markers was less affected by storage temperature under dry conditions than under wet conditions, while PAEP was detectable in samples stored below room temperature under wet conditions. The detectability of PAEP was considered to be the result of its higher expression level compared with MMP7 and LEFTY2. In conclusion, menstrual blood markers for the RT-qPCR procedure evaluated in this study were highly specific for menstrual blood. The proposed procedure could be useful for discriminating between menstruation and traumatic bleeding in the female genital tract. In particular, PAEP is expected to be applicable to forensic casework samples because of its high specificity and robustness.

The applicability of ELISA detection of gastric mucosa-expressing proteins for the identification of vomit.

The identification of vomit stains may be helpful for crime scene reconstruction. However, there is no specific and convenient method for identifying vomit stain. Therefore, to establish the procedure for forensic identification of vomit stains, we focused on four gastric mucosa-expressing proteins, pepsinogen I (PGA), pepsinogen II (PGC), gastrin (GAST), and mucin 5AC (MUC5AC). We developed enzyme-linked immunosorbent assay (ELISA) procedures for the detection of these four candidate proteins. The specificity and sensitivity of ELISA detection of these proteins were analyzed, and applicability for the identification of vomit in forensic casework samples was also investigated. We found the sensitivities of ELISA for detection of PGA, PGC, GAST, and MUC5AC from the standard protein (peptide) and from diluted gastric mucosa extract were 10.0-100.0 ng/ml and 1:200-1:1600, respectively. PGA and PGC were successfully detected in stomach contents and gastric mucosa samples; however, these also cross-reacted with some urine and semen samples, respectively, because of low level expression in these fluids. MUC5AC was positive for most gastric mucosa samples; however, it was difficult to detect in stomach contents. ELISA detection of GAST was not suitable for the identification of vomit. All aged samples stored up to 90 days gave positive results for ELISA procedures for PGA, PGC, and MUC5AC. Therefore, ELISA detection of these proteins might be applicable to aged samples. PGA was also detected in all actual vomit samples tested. These results suggest that ELISA for the detection of gastric mucosa-expressing proteins, especially PGA, could be an effective tool for the forensic identification of vomit.

Practical evaluation of an RNA-based saliva identification method.

Identifying saliva in samples found at crime scenes is important to clarify the tissue origin of DNA obtained for identification of individuals. Recently, a novel messenger RNA-based approach using two saliva-specific markers, Statherin (STATH) and Histatin 3 (HTN3), has been reported. This method can identify saliva more specifically than conventional amylase-based methods. Here, we performed several evaluations related to applying this method to real-world forensic work. First, we evaluated the effects of exposure to blue light (450nm) or to the reagent on Phadebas paper, which are direct methods used to locate saliva stains, on the stability of the RNA markers. The results demonstrate that exposure to the two direct tests did not affect the stability of the RNA markers. Second, we performed a comparative analysis of RNA-based and amylase-based conventional methods to examine the sensitivity and stability of the markers under various storage conditions. Although there was no difference in the sensitivity of the two methods for detecting 1-day-old saliva stains, a time-course study demonstrated that the RNA saliva markers were less stable than amylase, especially in wet conditions. During this time-course experiment, the stability of human DNA was also investigated. Although DNA was also unstable in wet conditions, it was more stable than the RNA markers in dry conditions. Taking the above results into consideration, we suggest that the RNA method could be introduced to current saliva identification procedures and should be used as a supplementary method to strongly support identification of saliva by the amylase-based method.

Identification and evaluation of potential forensic marker proteins in vaginal fluid by liquid chromatography/mass spectrometry.

Vaginal fluid is one of the most common body fluids found at crime scenes. Discriminating vaginal fluid from other body fluids is important in forensic science; however, few potential protein markers have been reported to date. Proteomic methods for identifying protein markers have gained attention, although few reports have applied this technology to forensic protein markers. Therefore, to identify characteristic vaginal proteins, we examined various body fluids (nasal secretions, saliva, urine, semen, vaginal fluids, and sweat) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry and peptide mass fingerprinting. We identified three components (average molecular mass values 17,237 ± 2, 18,063 ± 2, and 15,075 ± 1) detectable only in vaginal samples: two human small proline-rich protein 3 (SPRR3) isoforms and a human fatty acid-binding protein 5 (FABP5) with an acetylated (+42) N-terminal region lacking the initiator methionine residue (-131). Using ELISA, these yielded markedly high average values in vaginal fluids. The mass spectra of these proteins were not detected in infant saliva but were detected in the vaginal fluid throughout the menstrual cycle. The results of forensic analysis (detection limit, mixed body fluid samples, casework samples, and blind samples) suggest that these proteins are potential forensic markers. In conclusion, high SPRR3 and FABP5 expression levels, which may be used as potential markers for vaginal fluid identification in forensic science, were detected in vaginal fluids from healthy adults.

Effects on oral tissues of asphyxiation caused by cervical compression: The pink teeth phenomenon in Kato's studies (1941).

The effects on oral tissues of asphyxiation caused by cervical compression were investigated in 203 rabbits by Kiyotoshi Kato, who published four papers in classical Japanese in Kokubyo Gakkai Zasshi (now The Journal of the Stomatological Society, Japan) in 1941. The aim of this review is to summarize and disseminate the enormous amount of experimental data (reprint permission in English has been obtained from the journal). Based on the experimental models of hanging, strangulation with an external force equal to body weight, and strangulation with an external force equal to 1/10th body weight, the macroscopic and histological findings of the oral region immediately after death, including the respiratory condition until death and ocular conjunctival petechiae, were obtained and compared with 4 cases of human strangulation. Strangulation after each of four physical conditions (anesthesia, starvation, alcohol intake, and bleeding) was also investigated. Experiments were performed to investigate the effects of tracheal obstruction, cervical vein ligation, common carotid artery ligation, cervical nerve amputation, or combinations thereof. In the discussion focusing solely on tooth discoloration, it was suggested that the presence of pink teeth at a stage when putrefaction has little or no effect might be a finding indicative of death from asphyxiation caused by cervical compression. This review provides detailed data on the condition of oral tissues after asphyxiation caused by cervical compression and is expected to be used not only for elucidating the mechanism of the pink teeth phenomenon but also for conducting other forensic research.

Estimating the way of deposition of saliva stains using quantitative analysis of forensic salivary biomarkers.

Analyzing the way of deposition of saliva stains contributes to appropriate interpretation of saliva as evidence in court, particularly in sexual assault cases. In this proof-of-concept study, we aimed to confirm the difference between drooling-derived (non-contact) saliva and licking-derived (contact) saliva and clarify whether objectively distinguishing between the two saliva is possible. To allow discrimination between these two samples, an indicator was devised where the relative Streptococcus salivarius DNA quantity was calculated by dividing the S. salivarius DNA copies by the amount of stained saliva from the same saliva sample using quantitative polymerase chain reaction and salivary α-amylase activity assays. The study findings reveal that the value of the proposed indicator of licking-derived saliva was 100-fold significantly greater than that of drooling-derived saliva (P < 0.05, Welch's t-test). However, theoretical and technical challenges preclude the application of this indicator as a practical method. We believe that this saliva-specific bacterial DNA-based approach could allow estimation of the saliva stain deposition method.

Study on the Mechanism of the Pink Tooth Phenomenon Using Bovine Teeth: A Pilot Study.

The pink teeth phenomenon has occasionally been observed in forensic autopsies. This study aimed to establish an experimental pink tooth model and an objective color tone evaluation method in order to clarify changes in the color tone of teeth and the relationship with hemoglobin monoxide and its decomposition products and with red pigment-producing bacteria, under various external environmental factors. It was confirmed that the color tone evaluation with ΔE and the L*C*h color space was useful. The results of various examinations using this model showed that color development was suppressed under aerobic conditions, faded early under light, became bright red under a low temperature and showed a tendency to be reddish at 3 days under high humidity and in the presence of soft tissue. The biochemical analysis revealed a significant increase in carboxyhemoglobin at 7 days and a tendency toward increasing the total heme pigment and bilirubin levels over time. The bacteriological analysis revealed that red pigment-producing bacteria increased over time but that the color faded after 7 days. These results suggest that putrefaction greatly affects the pink teeth phenomenon, whereas red pigment-producing bacteria have little effect on the occurrence of pink teeth. However, further studies are needed to clarify bacteriological involvement.

Comparison of racemization rates between vital and endodontically treated teeth for age estimation.

Amino acid racemization of dentin (AAR) is among the most precise methods for age estimation in unidentified adult cadavers. Although vital teeth are generally used for this technique, cases often have endodontically treated (Endo) teeth only. Therefore, the aim of this preliminary pilot study was to determine the applicability of Endo teeth by comparing AAR rates between vital and Endo teeth. Thirty-six dentin samples from vital teeth and 18 dentin samples from Endo teeth were analyzed, including the maxillary first premolar, maxillary second molar, and mandibular second premolar. Standard calibration curves for the mandibular second premolar and maxillary second molar were compared among vital teeth, Endo teeth, and mixed vital/Endo teeth. Discrepancies between estimated and actual ages were assessed by analyzing the AAR rates for the maxillary first premolar between vital and Endo teeth. The AAR rates for Endo teeth were higher than those for vital teeth in both the mandibular second premolar and maxillary second molar, and the correlation of the standard calibration curve for vital teeth only was highest (r = 0.982836; r = 0.92011467), followed by mixed (r = 0.949579; r = 0.76158) and Endo teeth only (r = 0.896082; r = 0.744991). In the maxillary first premolar, discrepancies were more than 10 years for all 5 Endo teeth, based on the standard calibration curve of vital teeth. The present study suggests that the AAR rates for Endo teeth might be affected by variation in dentin structure between vital and Endo teeth.

Estimation of missing mandible from the skull using postmortem CT images.

Skeletal remains often have missing mandibles owing to the environment in which the corpse was placed or damage caused by an animal. Loss of the mandible reduces the accuracy of skull identification. Although several studies have validated methods for estimating mandibular morphology from conventional anthropological measurements using skull specimens, there are no reports using three-dimensional computed tomography (3DCT) images. Here, we examined methods for establishing the mandibular morphology from the remaining skulls using postmortem computed tomography (PMCT) images. We used PMCT images from 200 Japanese subjects as samples. After verifying the morphological correlation between the cranium and mandible, we created and validated estimation models using multiple regression analysis (stepwise method) for seven sites that were necessary for understanding the morphology of the mandible. Among the regression models, the estimated model for bicondylar breadth had the highest coefficient of determination (adjR2 = 0.53). We verified the accuracy of the model on a sample independent from the specimen used to create the estimation model and found that the formulated model of bicondylar breadth had good estimation accuracy, with a high correlation coefficient between the measured and predicted values of 0.82 and a mean absolute error of 3.582 mm, indicating that the model had good estimation accuracy. Here, we established a novel method for estimating the missing mandibular morphology based on PMCT data from the Japanese population. Our estimation model can help determine the missing mandibular morphology in a cranium remnant.

Usefulness of powdered crown of mandibular first premolars in birth year estimation by radiocarbon dating.

BACKGROUND: Radiocarbon (14C), whose levels increased in the atmosphere between 1955 and 1963, accumulates in the enamel of human teeth only during the process of tooth formation and has been applied to estimate the birth year of unidentified corpses. However, enamel isolation from teeth is time-consuming and labor-intensive. Therefore, this study aimed to estimate the birth year using 14C in the crown of a single mandibular first premolar tooth. MATERIALS AND METHODS: Fourteen mandibular first premolars collected from forensic autopsies were analyzed. For nine teeth, each tooth was cut longitudinally: half of the tooth was enamel, and the other half was crown (1). For the other five teeth, the entire crown was used (2). Thereafter, the 14C concentration in each tooth was measured using accelerator mass spectrometry. RESULTS: The absolute error between the actual date of birth (DOB) and the estimated DOB was within the range of 1.0-8.8 years in the enamel of five teeth and 0.1-4.0 years in the crown halves of seven teeth (1). For the five teeth with entire crowns, the absolute errors ranged between 0.3 and 3.0 years (2). CONCLUSION: The absolute error of 14C-based year of birth estimation using the powdered crown of the mandibular first premolar teeth ranged between 0.1 and 4.0 years. Our method, which involves pulverizing an entire crown, eliminates the need for the equipment, time, and labor associated with enamel isolation. Therefore, 14C dating using powdered crowns of mandibular premolars can be useful for birth year estimation.

Precise and comprehensive determination of multiple body fluids by applying statistical cutoff values to a multiplex reverse transcription-PCR and capillary electrophoresis procedure for forensic purposes.

Body fluid identification from crime scene evidence is an essential procedure in forensic investigations. Among various procedures, multiplex reverse transcription PCR assays have a clear advantage over conventional methods because different types of body fluids can be analyzed simultaneously. For more precise, comprehensive, and objective identification of forensically relevant body fluids, 15 target genes for blood, saliva, semen, vaginal fluid, and nasal secretion were selected; their primers were re-designed and multiplex PCR conditions were optimized to prioritize specificity for those body fluids. Multiple amplicons were separated and determined by the SeqStudio Genetic Analyzer with an all-in-one and easy-to-use cartridge. Then, the cutoff value was set for each marker to eliminate the detection of slight amplification in non-targeted body fluids. As a result, the targeted body fluid specificities of the developed procedure were drastically improved. Although successful determination of the target gene depends on sample condition and marker sensitivity, our procedure was applicable for the precise determination of body fluids in mixed body fluid stains, aged samples, and various mock casework samples. Therefore, it could be a powerful and convenient tool for the precise identification of multiple body fluids in forensic laboratories.

Forensic Characteristics of Physical Elder Abuse and Current Status and Issues of Collaboration between Forensic Medicine Departments and Related Institutions in Japan.

This study sought to clarify the characteristics and trends of physical elder abuse and the status of collaboration between forensic medicine departments and related institutions in Japan. Questionnaires were sent to 82 forensic medicine departments and 2857 institutions randomly selected from hospitals, municipalities and public community general support centers. The survey period was February to June 2021, including an extension period for collection. Responses from 675 facilities were analyzed. The most common finding in cases of physical elder abuse at forensic medicine departments was subcutaneous hemorrhage on the head (85.7%), with mixed old and new injuries most commonly observed in the lower limbs (70%). There were few cases in which there was collaboration between forensic medicine departments and other institutions. Among the issues identified, there is a need to provide related institutions with information obtained in forensic medicine departments. A new collaboration system is needed to achieve this.

Sixty-eight cases of postmortem pink teeth observed in dental autopsies of unidentified cadavers.

The pink teeth phenomenon was first reported in people who died by hanging or drowning in 1829. In recent years, there have been many reports that the occurrence of postmortem pink teeth is closely related to the condition of surroundings and time after death rather than to the cause of death, as had previously been thought. We observed pink teeth in 68 of 324 unidentified cadavers autopsied during 2015-2021, and we investigated the relationship of pink teeth with age, sex, cadaveric condition, time after death, tooth type, and cause of death. The results showed no significant differences in the occurrence of pink teeth according to sex or place of discovery (indoors, outdoors, or in water). Pink teeth tended to be observed below the age of 60 years. In the 68 cases with pink teeth, 29.1% of all vital teeth were affected, with the anterior teeth being most common. No evidence was found that supported an association between pink teeth and cause of death. These results are largely consistent with recent reports on postmortem pink teeth. As it stands, the pink teeth phenomenon may be nonspecific and must not be misinterpreted in medico-legal investigations. This case series provides further evidence supporting recent reports and contributes to clarifying in greater detail the mechanisms of the coloration and fading of pink teeth.

Validity of dental findings for identification by postmortem computed tomography.

Postmortem computed tomography (PMCT) images help identify individuals and extract information from corpses. PMCT may substitute for a standard examination when bodies are severely damaged or when resources are limited in a mass fatality incident. In such situations, the dental information revealed by PMCT has the potential to narrow down candidates for identification further. However, the validity of the dental findings obtained from PMCT images remains unclear. In this study, we investigated the validity of dental findings on PMCT images compared to regular dental examinations as the reference standard. We routinely collected PMCT images of 148 unidentified corpses and compared the dental findings of each tooth obtained from PMCT with those of a regular dental examination. The validity of dental findings of PMCT was measured by sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). We also evaluated the accuracy of dental findings per corpse using 32 teeth as the denominator. The dental findings with high sensitivity and high specificity were a present tooth (0.96 and 0.97), a missing tooth (0.97 and 0.97), pontic (0.92 and 1.00), a dental implant (1.00 and 1.00), and a root filling (0.94 and 0.99). The mean accuracy of the dental findings per corpse in PMCT was 95.6% (standard deviation: 6.9, minimum ≤ median ≤ maximum: 65.6 ≤ 100 ≤ 100). The number of corpses with 100% accuracy in the dental findings was 81 (54.7%). The information obtained in this study highlights the potential use of PMCT during human identification in several settings with limited resources, such as the number of specialists present and the condition of the corpses.

Oral bacterial DNA-based discrimination of human and canine saliva for the analysis of indistinct bite marks.

Analyzing ambiguous bite marks using conventional morphological approaches to identify attackers is difficult; thus, applying molecular biological methods for identifying an attacker from their saliva is a possible approach in a forensic investigation. This study aimed to establish oral bacterial DNA-based human and canine saliva markers and develop a practical method for their discrimination. We considered Streptococcus oralis and Pasteurella canis as human and canine saliva marker candidates, respectively. Duplex bacterial DNA detection using melting curve analysis was designed and evaluated for forensic applicability using proof-of-concept experiments. S. oralis DNA was detected from human saliva samples from 30 out of 30 individuals, and P. canis DNA was detected from canine saliva samples from 73 out of 77 individuals (26 dog breeds). Additionally, both bacterial DNA markers were accurately detected from human blood-contaminated saliva samples and mock indistinct bite marks. Our results indicate that both bacterial DNA markers were sensitive, robust, and discriminating saliva markers. We consider that our duplex bacterial DNA examination is a simple, practical, and useful method for the detection of saliva from indistinct bite marks and discrimination between human and canine saliva.

Fatal airway obstruction due to Ludwig's angina from severe odontogenic infection during antipsychotic medication: A case report and a literature review.

Ludwig's angina is characterized by inflammation of the sublingual and submandibular spaces and is mainly caused by odontogenic infection, which leads to cellulitis of the soft tissues of the floor of the mouth and the neck. This causes asphyxia due to elevation and posterior deviation of the tissues of the floor of the mouth. We report a fatal case of airway obstruction due to Ludwig's angina. A woman in her forties who had no physical complications, but had a mental illness, was undergoing outpatient dental treatment for caries in the first premolar of the left mandible. She was admitted to a psychiatric hospital because of insomnia caused by pain, where she developed cardiopulmonary arrest while sleeping and died 14 days after onset of the dental infection. Postmortem computed tomography (PMCT) prior to autopsy showed swelling of the soft tissues-from the floor of the mouth to the oropharyngeal cavity, the supraglottic larynx, and the prevertebral tissue. Autopsy revealed a markedly swollen face and neck, an elevated tongue, and a highly edematous epiglottis and laryngopharyngeal mucosa. There was also cellulitis and abscess of the facial, suprahyoid, and neck musculature, which suggested that the cause of death was asphyxiation due to airway obstruction. This was an alarming case, with mental illness leading to risk of severe odontogenic infection, and in which obesity and use of antipsychotic medication might have acted synergistically leading to airway obstruction. This is also a case of Ludwig's angina captured by PMCT, which has rarely been reported.

Detection of varicella-zoster virus DNA in 414 human trigeminal ganglia from cadavers by the polymerase chain reaction: a comparison of the detection rate of varicella-zoster virus and herpes simplex virus type 1.

Investigation of varicella-zoster virus (VZV) is important epidemiologically, and determination of its prevalence rate in human trigeminal ganglia is important to provide surveillance data. To date, studies on VZV detection in trigeminal ganglia have used specimens obtained from a relatively limited number of cadavers. This study attempted to detect VZV DNA as well as Herpes simplex virus type 1 (HSV-1) DNA by the polymerase chain reaction (PCR) from 414 samples of trigeminal ganglia obtained from 207 cadavers selected at random. The detection rate was examined to determine whether there were significant differences in the positive rate between the left and right trigeminal ganglia, males and females, and among age groups. A relationship was found between the positive rates for VZV and HSV-1. VZV DNA was detected in 391 of the trigeminal ganglia (94.4%) and 201 of the cadavers (97.1%) in 121/124 males and 80/83 females. HSV-1 DNA was detected in 251 of the samples (60.6%) and 134 of the cadavers (64.7%) in 72/124 males and 62/83 females. There was no significant difference for either virus in the detection rates between the left and right trigeminal ganglia and males and females. Age and positivity for HSV-1, but not VZV, showed a significant relationship. All 134 cadavers positive for HSV-1 were also positive for VZV. VZV and HSV-1 become latent in bilateral trigeminal ganglia, and are not affected by gender. The prevalence of HSV-1 was greater in advanced age, and the HSV-1-positive rate was correlated with the VZV-positive rate.

Determination of the geographical origin of unidentified cadavers based on geographical differences in genotype of varicella-zoster virus.

A new method was developed for determining the geographical origin of unidentified cadavers by examining the genome of varicella-zoster virus (VZV), which resides latently throughout life in most individuals and the genotypes which show distinct geographical distribution. VZV DNA samples extracted from the trigeminal ganglia of 62 cadavers (59 from Japan, and 1 each from the United Kingdom, Mongolia, and Pakistan) submitted for medico-legal autopsy were examined. Sequencing was performed on a 358-bp region in the open reading frame (ORF) 22 containing four single nucleotide polymorphisms (SNPs) and a 419-bp region in ORF 62 containing 2 SNPs in the VZV genome. Using these SNP markers, the VZV genome was classified into the nine genotypes described previously. Phylogenetic tree analysis was also undertaken for the sequenced regions and for the 22 existing VZV strains described previously. In addition, 21 samples were subcloned for detection of co-infection. The VZV genome was classified successfully into nine genotypes using four SNPs in ORF 22 and two SNPs in ORF 62 as markers. Among Japanese cadavers, 57 carried genotype J, 1 carried genotype M1, and 1 carried genotype M2. The British and the Mongolian cadavers carried genotype E1 and the Pakistani cadaver carried M1. Phylogenetic tree analysis showed that VZV strains can be classified into different genotypes with high bootstrap values. None of the subcloned samples showed evidence of co-infection. These results suggest that valuable additional information for determining the geographical origin of unidentified cadavers can be provided by examining the VZV genome.

Applicability of ELISA detection of statherin for forensic identification of saliva.

Statherin is a low molecular-weight phosphoprotein secreted from the parotid gland. Statherin mRNA was previously reported to be a useful marker for mRNA-based saliva identification. In this study, applicability of ELISA detection of statherin for forensic identification of saliva was investigated. The specificity and sensitivity of ELISA for detection of statherin were compared with those of ELISA for α-amylase and the Phadebas® amylase test. Statherin was specifically detected in saliva but not in other body fluids. In addition, statherin was successfully detected in aged saliva stains, mixed body fluids-saliva stains, and simulated casework samples. On the other hand, although ELISA for α-amylase showed higher sensitivity than ELISA for statherin, it was not specific enough to identify saliva. The Phadebas® amylase test also showed positive results in other body fluids that are known to have α-amylase activity; however, it is easy to use for screening forensic casework samples. In conclusion, ELISA for detection of statherin developed in this study could be an effective tool for the forensic identification of saliva because of its specificity for saliva among other body fluids. Forensic casework samples should be tested by ELISA detection or mRNA-based analysis for statherin, depending on the condition of the sample, to supplement presumptive tests for α-amylase, such as the Phadebas® amylase test.