Detection of bacterial 16S ribosomal RNA genes for forensic identification of vaginal fluid.

To preliminarily evaluate the applicability of bacterial DNA as a marker for the forensic identification of vaginal fluid, we developed and performed PCR-based detection of 16S ribosomal RNA genes of Lactobacillus spp. dominating the vagina and of bacterial vaginosis-related bacteria from DNA extracted from body fluids and stains. As a result, 16S ribosomal RNA genes of Lactobacillus crispatus, Lactobacillus jensenii and Atopobium vaginae were specifically detected in vaginal fluid and female urine samples. Bacterial genes detected in female urine might have originated from contaminated vaginal fluid. In addition, those of Lactobacillus iners, Lactobacillus gasseri and Gardnerella vaginalis were also detected in non-vaginal body fluids such as semen. Because bacterial genes were successfully amplified in DNA samples extracted by using the general procedure for animal tissues without any optional treatments, DNA samples prepared for the identification of vaginal fluid can also be used for personal identification. In conclusion, 16S ribosomal RNA genes of L. crispatus, L. jensenii and A. vaginae could be effective markers for forensic identification of vaginal fluid.

Evaluation of latex agglutination tests for fibrin-fibrinogen degradation products in the forensic identification of menstrual blood.

The identification of menstrual blood is important when discriminating menstruation from vaginal trauma in sexual assault cases. The aim of this study was to evaluate two fibrin-fibrinogen degradation product (FDP)-latex agglutination test kits, FDPL® Test (FDP-L) and FDP Plasma "RD" (FDP-P), for their ability to forensically identify menstrual blood. Sensitivity and specificity of the two kits were compared for menstrual blood and various body fluids, and the sensitivity of the FDP-latex agglutination test kit was also compared with that of an immunochromatographic test for human hemoglobin. The robustness of the FDP-latex agglutination test was compared with that of gene expression analysis of menstrual blood specific markers. The FDP-L kit was more sensitive than the FDP-P kit, but it cross-reacted with peripheral bloodstains from healthy volunteers. The FDP-P kit was specific for menstrual blood, with the exception of postmortem blood samples, and was not affected by other body fluids. In an FDP-negative menstrual blood sample, the sensitivity of human hemoglobin detection was lower than for FDP-positive samples and peripheral blood stains, suggesting that determination of human hemoglobin could be useful in interpreting negative results in the FDP-latex agglutination test. In menstrual blood samples incubated in wet conditions, FDP was found to be a robust marker in the identification of menstrual blood compared with mRNA markers. FDP-P testing was shown to be a suitable and highly efficient rapid screening test for the laboratory identification of menstrual blood.

Application of superimposition-based personal identification using skull computed tomography images.

Superimposition has been applied to skulls of unidentified skeletonized corpses as a personal identification method. The current method involves layering of a skull and a facial image of a suspected person and thus requires a real skeletonized skull. In this study, we scanned skulls of skeletonized corpses by computed tomography (CT), reconstructed three-dimensional (3D) images of skulls from the CT images, and superimposed the 3D images with facial images of the corresponding persons taken in their lives. Superimposition using 3D-reconstructed skull images demonstrated, as did superimposition using real skulls, an adequate degree of morphological consistency between the 3D-reconstructed skulls and persons in the facial images. Three-dimensional skull images reconstructed from CT images can be saved as data files and the use of these images in superimposition is effective for personal identification of unidentified bodies.

A novel method for ABO genotyping using a DNA chip.

ABO genotyping is often performed to identify the blood type of decomposed samples, which is difficult to be determined by a serological test. In this study, we developed a simple method for ABO genotyping using a DNA chip. In this method, polymerase chain reaction-amplified and fluorescent-labeled fragments in the ABO gene and primate-specific D17Z1 were hybridized with DNA probes on a chip designed to detect single nucleotide polymorphisms (SNPs) in the ABO gene and part of the D17Z1 sequence. Using blood samples from 42 volunteers and 10 animal species, we investigated whether the chip could be used to detect SNPs in the ABO gene and the D17Z1 sequence. This method was then applied to various forensic samples, and it was confirmed that this method was suitable for the simultaneous analyses of ABO genotyping and species identification. This method fulfills the recent need for the development of rapid and convenient methods for criminal investigations.

Application of postmortem 3D-CT facial reconstruction for personal identification.

Postmortem computed tomography (CT) images can show internal findings related to the cause of death, and it can be a useful method for forensic diagnosis. In this study, we scanned a ready-made box by helical CT on 2-mm slices in a mobile CT scanner and measured each side of the box to assess whether reconstructed images are useful for superimposition. The mean difference between the actual measurements and the measurements on the three-dimensional (3D) reconstructed images (3D-CT images) is 0.9 mm; we regarded it as having no effect on reconstruction for the superimposition method. Furthermore, we could get 3D-CT images of the skull, which were consistent with the actual skull, indicating that CT images can be applied to superimposition for identification. This study suggested that postmortem CT images can be applied as superimpositions for unidentified cases, and thinner slices or cone beam CT can be a more precise tool.

Evaluation of computed tomography as a screening test for death inquest.

The Japanese method of inquest, which depends mostly on external examinations, may misdiagnose a considerable number of accidental deaths and suicides as death by disease. We conducted computed tomography (CT) scans of 80 cases for which police concluded death by disease or natural causes based on police investigations into the circumstances and results from external examinations. The cause of death was clearly determined by CT scan in 17 of 80 cases. Ten cases underwent autopsy after the police suspected criminality based on results of the CT examinations. The results suggest CT scan may be a tool for preventing a number of overlooked crimes and accidents in Japan. However, it cannot be a perfect tool for discerning between death by disease and other causes of death without cooperation from the investigative agencies and subsequent forensic examinations such as autopsy and toxicological tests.

Two infant deaths linked to intussusception without peritonitis.

We report two infant deaths attributable to intussusception, but without clear evidence of peritonitis. In the first instance, a 3-year-old girl had presented with abdominal pain, vomiting, and melena before her demise. Aspirated vomitus was subsequently ascertained as the immediate cause of death, due to intussusception-induced ileus. The other infant, a 2-month-old male, showed autopsy evidence of intussusception at two sites, with likely aspiration of gastric mucus. Since the circumstances surrounding his death were vague, timing of the intussusception was difficult to pinpoint. Thus, an inconsequential, agonal event could not be discounted. Taken together, however, death from intussusception, without peritonitis, is the most viable postmortem interpretation for both patients. The causes of death in such cases are established by comprehensive delineation of preceding clinical events, plus autopsy documentation of coexistent intussusception and vomitus aspiration.

Evaluation of Tamm-Horsfall protein and uroplakin III for forensic identification of urine.

In this study, Tamm-Horsfall protein (THP), a major component of urinary protein, and uroplakin III (UPIII), a transmembrane protein widely regarded as a urothelium-specific marker, were evaluated for forensic identification of urine by ELISA and/or immunohistochemistry. THP was detected in urine, but not in plasma, saliva, semen, vaginal fluid, or sweat by the simple ELISA method developed in this study. In addition, most aged urine stains showed positive results. The urine specificity of THP was confirmed by gene expression analysis. Therefore, as reported previously, ELISA detection of THP can be used as a presumptive test for urine identification. UPIII was specific for immunohistochemical staining of cells in centrifuged precipitate of urine. However, ELISA and RT-PCR for UPIII were not specific for urine. UPIII may be applicable for forensic urine identification by immunohistochemistry.

Estimation of age from sclerotic glomeruli.

Glomerular sclerosis is one of the age-related causes of nephron damage. Histological studies of cadaver kidneys in several ethnic groups have shown that there is a consistent relationship between the percentage of sclerotic glomeruli (PSG) and age. However, no study regarding this relationship in the Japanese population has been reported to date. Here, we investigated such relationship in 150 Japanese cadavers that were selected regardless of clinical history. The straight line regression was estimated as follows: Age=23.3+1.36 x APSG (APSG: arcsine-transformed PSG). The R(2) value of the regression line was 0.598. The diagnostic test with the PSG value (cutoff=0.6%) for the age stratum (cutoff=33 years old) showed very high sensitivity (100%) and specificity (97.6%). From the results, PSG appears to be useful for the estimation of a cadaver's age in the Japanese population.

The relationship between cell membrane damage and lipid peroxidation under the condition of hypoxia-reoxygenation: analysis of the mechanism using antioxidants and electron transport inhibitors.

We consecutively observed lipid peroxidation and cell membrane damage under the condition of hypoxia-reoxygenation (H/R) in cells and analyzed their mechanisms by using electron transport inhibitors and an antioxidant. In H/R experiments, lipid peroxidation and cell membrane damage were observed during the hypoxia phase. In the reoxygenation phase, lipid peroxidation stopped, while cell membrane damage did not. An antioxidant, n-acetylcystein (NAC), and potassium cyanide (KCN) inhibited lipid peroxidation and cell membrane damage, while rotenone did not inhibit either of them. Although antimycin A did not inhibit lipid peroxidation, it inhibited cell membrane damage during the hypoxia phase but not during the reoxygenation phase. These results suggested that lipid peroxidation can affect cell membrane damage as a trigger during the hypoxia phase and the generation of oxidative stress can vary depending on the inhibition locations in the electron transport system.

Detection of Helicobacter pylori (H.pylori) DNA in digestive systems from cadavers by real-time PCR.

It is thought that there is a close relationship between regionality and DNA polymorphism of H.pylori. The application of H.pylori DNA to estimate the origin of unidentified cadavers may be possible. Previously, we detected H.pylori DNA in five stomach lesions by PCR in 50% of 100 cadavers in forensic autopsies. Furthermore, to see the localization of H.pylori in the digestive system, we tried to assay H.pylori DNA by real-time PCR. Mucous membranes at 14 points of digestive systems (5 points of the stomach, 1 point of the duodenum, 5 points of the small intestine, 3 points of the large intestine) were obtained from three cadavers whose stools were H.pylori-positive by an ImmunoCardST!HpSA detection kit. Genomic DNA was extracted using a DNA extraction kit. The 23Sr-DNA region was PCR-amplified (320 bp) by a TOYOBO Kit and the same region was assayed by real-time PCR using a Taqman probe. The PCR products were detected in 5 points of the stomach and 1 point of the duodenum in each sample. The more peripheral the intestine lesion was the weaker the band became. The quantitative PCR showed that there were more PCR products in 5 points of the stomach and 1 point of the duodenum than in the other tissues. H.pylori DNA was detected not only in the stomach but also in the duodenum and small intestine. There was a difference in the amount of H.pylori DNA detected in each organ. Further study is required for immunohistochemical staining, and to determine the relationship between a persons natural state and the H.pylori DNA polymorphism (which codes for CagA).

Multiplex STR typing of aortic tissues from unidentified cadavers.

The DNA of aortic tissues collected at the autopsies of unidentified 47 cadavers was examined using a multiplex short tandem repeat (STR) typing kit. The causes of death included drowning, burning and brain injury among others. Tissues samples were stored in ethanol before DNA extraction. DNA was extracted from about 25mg of dried tissues using a Q1Amp DNA Mini kit (QIAGEN). STR typing was performed using an AmpFlSTR Identifiler PCR Amplification kit (Applied Biosystems) and GeneMapper ID software v. 3.2 (Applied Biosystems). The amount of recovered DNA ranged from 0.006 to 3.44 microg/mg. Tissue samples were collected at estimated times between 1 day and 2 years after death. We were able to type 46/47 tissue samples (98%) and all 15 STR alleles and the amelogenin gene were detected in 38 cases (81%). Successful typing was completed for most tissue samples taken less than 1 month and up until 3 months after death. As the days after death increased, the numbers of alleles with longer DNA fragment sizes decreased. These results suggest that the DNA from aortic tissues can be accurately typed for multiplex STR and amelogenin until about 1 month after death. We found that aortic tissues are one of the most useful samples for forensic personal identification of unidentified bodies.

Can cervical spine injury be correctly diagnosed by postmortem computed tomography?

We discuss the usefulness of postmortem computed tomography (PMCT) by reviewing cases of cervical spine injury. A merit of PMCT is that it can identify injury that cannot be found on autopsy; however, peculiar defects of it may exist. While PMCT can identify bone fractures, it cannot indicate whether the injury was inflicted while the deceased was still alive or not because of its inability to clearly image bleeding around the fracture. Furthermore, CT often misses some types of cervical spine injuries, such as laceration of an intervertebral disk and incomplete fracture of the cervical spine. On the other hand, cervical spine injury on CT images occasionally has an appearance similar to subarachnoid hemorrhage due to rupture of the cerebral artery, indicating that cervical spine injury can be misdiagnosed as a disease by PMCT. When PMCT is used for screening trauma, caution must be observed regarding its limitations. If the possibility of trauma of the neck or head is not completely ruled out from the personal history of the victim, autopsy is strongly recommended, even when PMCT findings indicate that the cause of death may be due to disease, such as subarachnoid hemorrhage.

Forensic application of Epstein-Barr virus genotype: correlation between viral genotype and geographical area.

Using 50 forensic blood samples, the latent membrane protein 2A (LMP-2A) gene of Epstein-Barr virus (EBV) DNA was amplified to find a geographic correlation among the EBV genotypes. EBV DNA was detected in nine samples. From a phylogenetic analysis using 18 reported sequences as a reference, six EBV subtypes (Ia, Ib, Ic IIa, IIb, and IIc) were found. Japanese isolates were included in subtypes Ia or IIa. All the Asian reference isolates, except isolate D6, were included in subtype Ia or IIa. Mediterranean, an Alaskan and other African isolates were included in types Ib, Ic, IIb and IIc. The EBV genotype in the LMP-2A gene was thus demonstrated as being correlated with the host's geographical location. Typing in the EBV-associated nuclear antigen 2 gene was not related to that in the LMP-2A gene. Detection of the EBV genotype in the LMP-2A gene may be useful for determining the geographical origins of unidentified cadavers.

A fatal case of hypovolemic shock after cesarean section.

We report a fatal case of hypovolemic shock caused by uncontrollable hemorrhaging after emergency cesarean section. In this patient, the incision in the uterus was located only 1 cm from the cervical os. We suspect that this close incision was the cause of the damage to the uterine venous plexus and the bleeding. We discuss the cause of death and offer advice on performing autopsies in patients who have died of bleeding after cesarean section.

BK virus genotype distribution offers information of tracing the geographical origins of unidentified cadaver.

There are no efficient methods to determine the geographic origin of unidentified cadavers. We showed earlier that the geographical distribution of the JC virus genotype detected from human kidneys indicates the host's geographical origin. As there are still cadavers from which we cannot detect the JC virus (JCV), we investigated whether the genotype of another virus species belonging to the same family, human BK virus (BKV), could also be used to detect human geographical origin. BKV was found in 11 of 36 cases (30.5%). Even in the seven JCV-negative cases, the host's geographic origin could be estimated from the BKV genotype. Four subjects were positive for both the BKV and JCV. As the distribution areas of BKV and JCV genotypes are not identical, it is possible to narrow down the geographic area that any cadaver originates from.

Detection of herpes simplex virus type 1 DNA in bilateral human trigeminal ganglia and optic nerves by polymerase chain reaction.

Herpes simplex virus type 1 (HSV-1) DNA was extracted from human trigeminal ganglia of 121 cadavers aged between 3 months and 94 years, and its PCR amplification was performed for the RL2 HSV-1 sequence using two pairs of primers. The HSV-1 DNA was detected in 74 of 121 patients (61%); 70/74 bilaterally, 3/74 only on the left side, and 1/74 only on the right side. Although the PCR-positive rate was significantly higher in advanced age, the correlation between the PCR-positive rate and gender was unclear. Additionally, HSV-1 DNA was not detected in any of the 50 optic nerve samples.

Usefulness of dura mater in providing DNA samples for identifying cadavers.

We examined the usefulness of the dura mater in identifying human remains. Dura mater was collected from 50 cadavers, including drowned, charred, and mummified remains. The STR genotype using the AmpFlSTR Identifiler Kit could be typed at 15 STR and amelogenin loci in 30 samples of 33 cases. Furthermore, the ABO genotype and amelogenin using gel-based methods could be typed in 44 samples of 50 cases. In cases with successful typing of STR, ABO-DNA, and amelogenin, the longest time after death was from 12 to 26 days in a drowned body. The minimum quantity of dura mater required for DNA extraction was about 2.5 mg, dried and fixed by ethanol, in a cadaver 15 h after death. The state of the DNA from the dura mater from the calvaria may be better than that from the basis cranii interna. We found that DNA from dura mater is one of the most useful samples for forensic identification.

Ethanol production by Candida albicans in postmortem human blood samples: effects of blood glucose level and dilution.

We present two cases in which the ethanol concentration in blood samples taken after death continued to increase in the absence of any remarkable increase in n-propanol concentration. Species of bacteria and yeasts, including Candida albicans were isolated from these samples. We then examined whether C. albicans, the most common yeast in the general environment, was able to produce ethanol in human blood stored at room temperature. Ethanol production increased as the glucose concentration increased, indicating that C. albicans produced ethanol from the glucose. Our results also suggested that C. albicans produced ethanol more easily in blood diluted by intravenous infusions that included glucose than in undiluted blood. These findings are useful for the evaluation of postmortem ethanol production in subjects whose blood has been diluted by infusions with glucose. Furthermore, there was no quantitative relationship between the amount of n-propanol detected and the amount of ethanol production: n-propanol appears to be an unreliable index of putrefaction and postmortem ethanol production by C. albicans. It is possible for the blood ethanol level to be high and n-propanol not to be detected, even if the subject has not been drinking alcohol. We reconfirmed the necessity of immediately adding sodium fluoride to samples for ethanol analysis to prevent postmortem ethanol production.

Does imaging technology overcome problems of conventional postmortem examination? A trial of computed tomography imaging for postmortem examination.

We used a mobile computed tomography (CT) unit for postmortem examinations of deceased subjects to see how many mistakes on cause-of-death diagnoses were made in Japan. In 5 of 20 cases, the cause of death determined by CT was different from the diagnosis made by superficial postmortem examination. In one case, the superficial examination suggested no trauma, whereas a subdural hematoma was found on cranial CT images. We concluded that postmortem examinations in Japan were not effective when screening for crimes or accidents. Using a mobile CT scanner in postmortem examination may be a viable method of screening for causes of deaths, although it cannot be used as a substitute for autopsy.