Usefulness of Heart-Type Fatty Acid-Binding Protein Measurement in Postmortem Urine Specimens.

ABSTRACT: Heart-type fatty acid-binding protein (HFABP) is a 15-kDa substance reported to pass through the renal tubules and be renally excreted. Therefore, it is possible that its concentration in the urine collected postmortem may reflect antemortem blood levels. We measured the postmortem urine concentration of HFABP in 94 forensic autopsy cases and compared it between acute myocardial infarction (AMI), sepsis, heat stroke cases, and asphyxia cases as control cases to examine its diagnostic validity. Kidney tissue collected at autopsy was immunostained with antibodies against HFABP to evaluate the correlation with the urinary measurements. Urinary HFABP was significantly higher in AMI, sepsis, and heat stroke cases than in asphyxia cases. Quantitative immunostaining results showed no significant differences between any 2 groups. The usefulness of kidney immunostaining for HFABP in elucidating the cause of death was low. Two reasons may explain the lack of significant differences in kidney immunostaining: nonspecific leakage of tubular epithelial HFABP into the tubules because of postmortem changes and oliguria due to dehydration caused by heat stroke. In conclusion, the measurement of urinary HFABP may be useful in elucidating the cause of death; however, the kidney HFABP immunostaining was not significantly different from AMI.

Ruptured Aortic Valve Aneurysm Caused by Infective Endocarditis in a SARS-CoV-2-Positive Autopsy Case.

ABSTRACT: Aortic valve aneurysm, an extremely rare complication secondary to infective endocarditis (IE), may cause heart failure due to rupture of the aneurysm. Coronavirus disease 2019 (COVID-19) has been reported to cause cardiovascular complications and alter susceptibility to secondary infections such as IE. Herein, we report a case of IE with a fatal outcome caused by rupture of an aortic valve aneurysm in a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive autopsy case. The patient was a 67-year-old male diagnosed with heart failure after presenting with edema and weight gain. He was found dead in bed 2 weeks after initial symptom presentation. Autopsy revealed an aneurysmal sac in the center of the noncoronary cusp of the aortic valve with an opening of approximately 1 cm in the center of the aneurysmal wall. Histologically, aortic valve vegetation, destruction of the aortic valve cusp, rupture of the aneurysmal wall, and an abscess under the aortic intima were observed. Gram staining of the aneurysmal wall showed a gram-positive coccus. The reverse transcription quantitative polymerase chain reaction assay was positive for SARS-CoV-2. Because no defined risk factors for IE other than SARS-CoV-2 infection were observed, the association between IE and COVID-19 was highly likely.

Prominent black esophagus, Wischnewsky spots, and black duodenum in a fatal hypothermia case with an underlying diabetic ketoacidosis.

Hypothermia can occur in patients with diabetic ketoacidosis (DKA), and these two conditions can exacerbate each other. Fatal hypothermia and DKA have overlapping features and findings such as Wischnewsky spots (WS), black esophagus, basal subnuclear vacuolization in the renal tubule, dehydration, and increased acetone levels. Therefore, it may be challenging to differentiate or clarify the context of these two conditions. Herein, we report a case of a 49-year-old man with type 1 diabetes who was found lying in his house in mid-winter. He experienced cardiopulmonary arrest 10 h after the initial discovery and died at the hospital. On autopsy, florid left cardiac blood was observed. Black discoloration of the distal part of the esophageal mucosa, widespread WS in the gastric mucosa, and black discoloration of the duodenal mucosa were observed. Histologically, neutrophil infiltration in the esophageal mucosa, neutrophil infiltration and bleeding in the gastric mucosa, basal subnuclear vacuolization and Armanni-Ebstein lesion in the renal tubule epithelium in the kidney, and hyalinization of the islets of Langerhans were observed in the pancreas. Blood acetone and ß-hydroxybutyrate levels were 538 µg/mL and 8947 µmol/L, respectively. Glycated hemoglobin A1c and glucose levels were 16.2% and 883 mg/dL, respectively, while C-reactive protein level was 3.64 mg/dL. In conclusion, obnubilation due to DKA was assumed to be the underlying cause of hypothermia, and the combination of these two conditions led to the outcome of death. The concurrent presence of these conditions likely contributed to the conspicuous mucosal findings in the upper gastrointestinal tract.

Polymorphism and haplotype analysis of three novel short tandem repeat loci in the p11.4 region of human X chromosome.

X-chromosomal short tandem repeats (X-STRs) are useful for the identification of absent single parents and complex blood relations. In the present study, we aimed to identify novel STR loci for use as DNA markers by conducting polymorphism and haplotype analyses. We detected three novel STR loci (LC552061, LC552062, and LC552063, with repetitive structures of (GGAA)n(GGGA)m, (CCTT)n(CCCT)m, and (ATTT)n, respectively) in the p11.4 region of the X chromosome. For these X-STRs, the polymorphism information content values ranged from 0.5766 to 0.6377 and the power of discrimination in males and females ranged from 0.6269 to 0.6844 and from 0.8105 to 0.8537, respectively. The linkage disequilibrium analysis revealed p values of < 0.0001, < 0.0001, and 0.00909 between LC552061 and LC552062, LC552061 and LC552063, and LC552062 and LC552063, respectively. Additional linkage disequilibrium analysis including seven previously analyzed loci (LC149476, LC149479, LC149480, LC149484, LC317283, LC317284, and LC317285) revealed a p value of < 0.001 among each of the five loci (LC149476, LC149479, LC149480, LC149484, and LC317283) and between LC317284 and LC317285, indicating that they were a linked group. These results indicate that, in addition to the seven previously detected loci, the three novel X-STR loci identified in the present study might be useful DNA markers for complex kinship analysis and might support the Investigator® Argus X-12 kit.

Accuracy of Urea Nitrogen and Creatinine Measurements in Postmortem Serum and Pericardial Fluid Compared With Antemortem Data.

ABSTRACT: Although several studies have measured urea nitrogen (UN) and creatinine (Cr) concentrations in postmortem serum and pericardial fluid, no recent antemortem biochemical data have been available for forensic autopsy, thereby making the evaluation of the accuracy of postmortem data difficult. This study compared antemortem (from emergency room results before the declaration of death) and postmortem serum UN and Cr concentrations, as well as postmortem serum and pericardial fluid values, in 51 forensic autopsy cases (postmortem interval within 87 hours). Postmortem UN concentrations were strongly correlated with antemortem data. Moreover, no significant difference between pericardial fluid UN concentrations and antemortem data was observed. Postmortem serum and pericardial fluid Cr values were also correlated with antemortem data, although postmortem values were significantly higher than antemortem ones. Given our observation of early postmortem elevation in Cr concentrations, such an elevation was attributed to rigor mortis. In conclusion, the current study demonstrated the utility of postmortem UN and Cr concentrations, in particular of those measured in the pericardial fluid.

Comprehensive Severe Acute Respiratory Syndrome Coronavirus 2 Detection Using Polymerase Chain Reaction and Rapid Antigen Testing in Postmortem Specimens.

ABSTRACT: Polymerase chain reaction (PCR) is indispensable for diagnosing coronavirus disease 2019 (COVID-19) in autopsy cases. In this study, we performed comprehensive reverse transcription quantitative PCR (RT-qPCR) and rapid antigen tests for COVID-19 on forensic postmortem specimens, regardless of the antemortem symptoms and causes of death. Immediately before forensic external examination and autopsy, a wiping solution was collected from the nasopharynx with a dry swab, and rapid antigen testing and RT-qPCR were performed. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected by RT-qPCR in 12 of the 487 cases; the infection rate was 2.46%. Of the RT-qPCR-positive cases, 7 were associated with COVID-19-related deaths. Cycle threshold values were not correlated with the cause of death or postmortem time. The sensitivity and specificity of the rapid antigen test were 91.67% and 100.00%, respectively. The RT-qPCR positivity rate of forensic cases was higher than the cumulative infection rate for the entire population. SARS-CoV-2 could be detected with the rapid antigen test and RT-qPCR within 216 hours of death. Because the rapid antigen test showed the same sensitivity and specificity as those observed in clinical practice, the test combined with RT-qPCR may be useful for diagnosing COVID-19 even in postmortem specimens.

Effectiveness of Rapid Antigen Testing in Forensic Cases of Severe Acute Respiratory Syndrome Coronavirus 2 Infection, Including Delta Variant.

ABSTRACT: The polymerase chain reaction is indispensable for diagnosing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in forensic cases. However, studies regarding the effectiveness of rapid antigen testing (RAT) in forensic cases remain limited. Therefore, we investigated the efficacy of RAT compared with reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for confirming SARS-CoV-2 infection (including the delta variant). Before the external examination or autopsy, we collected samples from the nasopharyngeal mucosa, which were then assessed via RAT (QuickNavi COVID-19 Ag kit, QuickNavi-Flu+COVID-19 Ag kit) and RT-qPCR. Reverse-transcription quantitative polymerase chain reaction results were positive in 73 of 1255 cases, and 21 cases were identified as those of delta variants. Low RT-qPCR threshold cycle value cases and delta variant infections were more likely to result in coronavirus disease-related deaths. The sensitivity of the QuickNavi COVID-19 Ag kit was 76.32%, and that of the QuickNavi-Flu+COVID-19 Ag kit was 77.14%. The specificity of both RATs was 100%. In QuickNavi COVID-19 Ag kit cases, delta variant cases showed lower sensitivity than non-delta variant cases, even for a similar viral load. Thus, RAT in forensic cases is sufficiently useful as a screening test for SARS-CoV-2 infection. However, RAT carries a risk of false negatives, especially for delta variant cases.

Cerebral venous sinus thrombosis associated with COVID-19: an autopsy case report.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the SARS-CoV-2 virus. COVID-19 has been reported to increase the propensity for systemic hypercoagulability and thromboembolism disorders such as cerebral venous sinus thrombosis (CVST). A 66-year-old woman was found dead at her home. She had symptoms of fever, dizziness, and malaise 2 weeks prior to her death. However, her fever declined 3 days before death. Postmortem computed tomography conducted before the autopsy suggested CVST. On autopsy, a massive thrombus was observed from the cortical veins to the superior sagittal sinus and transverse sinus accompanied by a small infarction region in the left parietal region. Although the rapid antigen test was negative, the reverse transcription-quantitative polymerase chain reaction test was positive for SARS-CoV-2, with a cycle threshold (Ct) value of 38.9. The serum C-reactive protein level was 0.532 mg/dL. COVID-19 was the only risk factor for CVST, and no other cause of death was determined. Therefore, the cause of death was determined as acute intracranial hypertension due to CVST associated with COVID-19. The patient died after the symptoms improved, the Ct value of RT-qPCR was 38.9, and the serum C-reactive protein level decreased. Therefore, CVST might have occurred in the convalescent phase of COVID-19 infection.

Rescue from Stx2-Producing E. coli-Associated Encephalopathy by Intravenous Injection of Muse Cells in NOD-SCID Mice.

Shiga toxin-producing Escherichia coli (STEC) causes hemorrhagic colitis, hemolytic uremic syndrome, and acute encephalopathies that may lead to sudden death or severe neurologic sequelae. Current treatments, including immunoglobulin G (IgG) immunoadsorption, plasma exchange, steroid pulse therapy, and the monoclonal antibody eculizumab, have limited effects against the severe neurologic sequelae. Multilineage-differentiating stress-enduring (Muse) cells are endogenous reparative non-tumorigenic stem cells that naturally reside in the body and are currently under clinical trials for regenerative medicine. When administered intravenously, Musecells accumulate to the damaged tissue, where they exert anti-inflammatory, anti-apoptotic, anti-fibrotic, and immunomodulatory effects, and replace damaged cells by differentiating into tissue-constituent cells. Here, severely immunocompromised non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice orally inoculated with 9 × 109 colony-forming units of STEC O111 and treated 48 h later with intravenous injection of 5 × 104 Muse cells exhibited 100% survival and no severe after-effects of infection. Suppression of granulocyte-colony-stimulating factor (G-CSF) by RNAi abolished the beneficial effects of Muse cells, leading to a 40% death and significant body weight loss, suggesting the involvement of G-CSF in the beneficial effects of Muse cells in STEC-infected mice. Thus, intravenous administration of Muse cells could be a candidate therapeutic approach for preventing fatal encephalopathy after STEC infection.

Analysis of four novel X-chromosomal short tandem repeats within 71 kb of the Xp22.3 region.

DNA testing using X-chromosomal short tandem repeat (X-STR) polymorphisms has been used in maternity/paternity and complex kinship cases. Analyses of repeat sequences, surveys on racial statistics, and development of practical applications for DNA testing continue to be reported. In this study, we identified four novel tetranucleotide STR loci located in the X chromosome, which is the basis of X-STR research. These four tetranucleotide STRs were located within 71 kb of the chromosome Xp22.3 region. Using sequence analysis of the structure of repeat sequences, we identified simple repeat sequences of TAAA, CTTT, TATC, and GATA with rare insertions. We then calculated forensic statistical parameters using base length analysis. In the Japanese population, the polymorphism information content was 0.597-0.687, power of discrimination in females was 0.829-0.884, and power of discrimination in males was 0.635-0.729. As these tetranucleotide STRs are closely linked, we conducted haplotype analysis and detected that three loci (LC149476, LC149479, and LC149480) were in linkage disequilibrium. We demonstrated that the simultaneous analysis of these loci may be useful in complex kinship cases. Because these four loci can be detected by multiplex PCR, the detection of alleles at these loci can be rapidly and easily achieved. We conclude that the X-STR loci detected in this study may be useful tools in complex kinship cases and may increase the reliability of genetic testing.

Immunohistochemical analysis of the ubiquitin proteasome system and autophagy lysosome system induced after traumatic intracranial injury: association with time between the injury and death.

This study aimed to analyze how the ubiquitin proteasome system (UPS) or autophagy lysosome system (autophagy) are induced in brain tissues at different intervals after traumatic intracranial injury in humans. Injured cerebral cortices of 36 forensic autopsy cases were analyzed by immunohistochemistry using antibodies as the UPS marker (ubiquitin and lysine 48-linked polyubiquitin chains [K48]) and autophagy marker (lysine 63-linked polyubiquitin chains [K63], p62/sequestome 1 and microtubule-associated protein 1 light chain 3 [LC3]). The number of neurons and glial cells with cytoplasmic inclusions that stained positive for ubiquitin, K48, and p62 began to increase within 1 hour after intracranial injury, particularly at contusion sites. From 3.5 hours onward, an increase in cytoplasmic inclusions that stained positive for K63 and LC3 began to be detected. LC3-positive cytoplasmic inclusions were not identified after 37 days; however, the increased immunoreactivity to ubiquitin and anti-K48 antibody was maintained for 7 months. These results suggest that the UPS is activated earlier and lasts longer than autophagy, that autophagy is activated for a relatively short term (between a few hours and approximately 1 month), and that the activation occurs especially in severely damaged brain tissues following head trauma in humans.

A fatal fall associated with undiagnosed parenchymatous neurosyphilis.

A fall from a great height is often associated with altered mental status. Aside from the usual contributing factors, for example, alcohol consumption or mental illness, natural disease leading to a fatal fall is seldom identified by autopsy. The case described in this report is that of a 57-year-old man who had been clinically diagnosed with presenile Alzheimer disease and fell head first into a river from the bridge. These events were captured by a surveillance camera on the bridge; an acquaintance reported that he had previous suicidal ideation. At autopsy, the cervical spinal cord was determined to have been severely injured, and the sixth cervical vertebra was observed to have been fractured. Histological examination showed chronic meningoencephalitis including neuronal loss, perivascular cuffing, and the proliferation of microglia and astrocytes in the cerebral cortex. Serologic evaluation consisting of the nontreponemal antigen test (rapid plasma reagin [RPR]) and treponemal antibody test (Treponema pallidum latex agglutination) was positive in both cases (RPR: 18.2 RPR units, T. pallidum latex agglutination: 7718 U/mL). These findings suggested that the patient had been affected by a syphilis infection and that his suicidal behavior was associated with parenchymatous neurosyphilis.

The influence of circulating blood volume on blood ethanol concentrations in a rat model.

In the medicolegal field, whether a victim was under the influence of ethanol at the time of an accident or injury is an important issue. However, trauma victims may have lost a large amount of blood, or received cardiopulmonary resuscitation or infusions of fluid or blood, making it difficult to interpret the ethanol concentration at the time of sampling. We, therefore, investigated changes in ethanol elimination in a rat model in which variations in circulating blood volume were induced by means of slow hemorrhage or infusion. Animals were divided into four experimental groups: hemorrhage (H), infusion (I), hemorrhage and infusion (H&I), and control (C). All rats were administered ethanol (1 g/kg body weight) intravenously over a period of 5 minutes. Blood (group C and group I: 0.2 ml, group H and group H&I: 0.5 ml/300gBW) was collected from rats in each group every 10 minutes for 4 h. Every 10 minutes after 30 minutes to 4 h of the ethanol administration, the rats in the infusion groups were administered saline (group I: 0.5 ml/300gBW, group H&I: 1.0 ml/300gBW). The concentration of ethanol in the blood samples was determined by using head-space gas chromatography. We found that the ethanol elimination rate did not differ between the groups, indicating that variations in body fluid due to bleeding or infusion have little to no effect on blood ethanol concentrations. Blood ethanol levels obtained after a prolonged state of shock or agonal, however, may need to be cautiously interpreted.

Relationship between N-terminal pro-brain natriuretic peptide concentration and heart-type fatty acid-binding protein in postmortem urine.

The clinical use of N-terminal pro-brain natriuretic peptide (NT-proBNP) and blood concentrations of heart-type fatty acid-binding protein (HFABP) is well-established in diagnosing heart conditions. However, their applicability in forensics is controversial due to postmortem changes. NT-proBNP and HFABP are excreted in the urine due to their small molecular weights and may be found in postmortem urine samples; however, their correlation has not been evaluated. In this study, we compared the concentrations of urinary NT-proBNP and HFABP in 386 forensic autopsy cases. The urinary NT-proBNP levels were significantly higher in acute myocardial infarction (AMI), congestive heart failure (CHF), sepsis, and hyperthermia cases, with the highest levels in CHF cases. Similarly, HFABP concentration was significantly higher in CHF, sepsis, and hyperthermia cases, with the highest level observed in hyperthermia cases. However, the difference in urinary HFABP levels between the AMI and control cases was not significant. Our analysis revealed a correlation between postmortem urine NT-proBNP and HFABP levels, and the NT-proBNP/HFABP ratio was high in patients with CHF and sepsis cases and low in those with hyperthermia. The difference between the ratios was possibly due to the combined release of ventricular myocardial cells in response to ventricular wall stress and myocardial injury for NT-proBNP, as well as myocardial and skeletal muscle injuries for HFABP. This study, for the first time, demonstrates the utility of postmortem measurements of urinary NT-proBNP and HFABP levels, offering valuable insights for improving the accuracy of postmortem diagnosis in forensic medicine.

Beading of the astrocytic processes (clasmatodendrosis) following head trauma is associated with protein degradation pathways.

OBJECTIVES: To present the incidence of clasmatodendrosis (injured astrocytic processes) and analyse the association between clasmatodendrosis and the ubiquitin proteasome system (UPS) and autophagy lysosome system (autophagy). METHODS: The injured cerebral cortices of 36 autopsy cases were analysed by light microscopy by immunohistochemistry using an anti-glial fibrillary acidic protein (GFAP) antibody and UPS marker antibodies (ubiquitin and lysine 48-linked polyubiquitin chains (K(48)) and autophagy marker (lysine 63-linked polyubiquitin chains (K(63)), p62/sequestosome 1 and microtubule-associated protein 1 light chain 3 [LC3]). Double-immunofluorescence staining was used to detect the relationships between p62-K(48), p62-K(63) and GFAP-K(48). RESULTS: Clasmatodendrosis that was immunoreactive to GFAP or K(48) was detected by 1 hour up to 14 days following head trauma. The survival time of cases with clasmatodendrosis was significantly shorter than that in cases without clasmatodendrosis (p < 0.01). Involvement of contusion or oedema was significantly frequent in cases with clasmatodendrosis (p < 0.01). There was no significant age difference between cases with clasmatodendrosis and cases without clasmatodendrosis (p = 0.18). Double-immunofluorescence staining for p62-K(48) and GFAP-K(48) demonstrated clasmatodendrosis with overlapping fluorescence signals. CONCLUSION: Clasmatodendrosis is associated with UPS-mediated, autophagy and relatively acute pathological findings after traumatic intracranial injury.

An experimental study on the macroscopic findings of ligature marks using a murine model.

Macroscopic findings of ligature marks are mainly affected by the characteristics of the ligature, the severity of external forces acting on the ligature, and the period that the neck has been pressed by the ligature. Therefore, the appearances of ligature marks formed by ligatures with the same characteristics differ depending on 2 factors: force and time. To examine which of these factors contributes more significantly to the macroscopic findings of ligature marks, a semiquantitative and experimental study using a murine model was performed. We experimentally made artificial ligature marks by hanging different sized weights using a vinyl band on dehaired legs of rats for different periods, both intravitally and postmortemly.After weights of 1, 3, and 6 kg with vinyl bands were hung on the legs of rats for 0 to 24 hours, the depths of ligature marks were semiquantitatively evaluated in 4 grades. Macroscopic examination and statistical analysis revealed that the appearance of ligature marks is not affected by whether they are formed intravitally or postmortemly but that it is equally affected by the severity of force and the duration of force application. We believe that the results of this study will be helpful to determine the characteristics of ligature and the period of hanging or strangulation time in the practice of forensic medicine.

Postmortem pericardial fluid sLOX-1 levels and LOX-1 immunostaining in forensic specimens: Relation to cause of death.

Lectin-like oxidized LDL receptor-1 (LOX-1) is the endothelial receptor for oxidized LDL. This receptor's extracellular domain is released into the blood as soluble LOX-1 (sLOX-1) and has been linked to ischemic heart disease (IHD), cerebrovascular diseases (CVDs), obesity, and diabetes. We recently reported that sLOX-1 fluid levels in postmortem pericardial fluid were comparable to clinical values in live patients and that significant increases in sLOX-1 were observed in patients with IHD. However, postmortem serum and urine sLOX-1 levels were higher than serum levels in living patients. Here, we conducted LOX-1 immunostaining in forensic specimens (aorta and heart) and evaluated pericardial fluid sLOX-1 in 221 medicolegal autopsy cases (67 IHD, 11 CVD, 17 inflammatory diseases, and 126 control cases) with a postmortem interval < 72 h to assess the diagnostic efficiency of postmortem pericardial fluid sLOX-1. Furthermore, we evaluated the relationships between pericardial fluid sLOX-1 and body mass index (BMI), blood HbA1c, serum C-reactive protein (CRP), high-density lipoprotein cholesterol (HDL-C), and low-density-lipoprotein cholesterol (LDL-C). LOX-1 immunostaining positivity was found in the aortic intima. Pericardial fluid sLOX-1 levels were considerably higher in patients with IHD and CVD. However, there were no significant differences in patients with inflammatory diseases and controls. No associations between pericardial fluid sLOX-1 and BMI, HbA1c, CRP, HDL-C, or LDL-C were found. These results indicate sLOX-1 utility in the postmortem diagnosis of IHD and CVD.

Molecular genetic analysis of nongestational choriocarcinoma in a postmenopausal woman: a case report and literature review.

Choriocarcinoma is a highly malignant tumor of trophoblastic origin. Most cases occur in association with preceding gestational events. However, on very rare occasions, nongestational choriocarcinoma arises from germ cell or trophoblastic differentiation in different types of carcinoma. This article reports the case of a 58-year-old woman with primary nongestational choriocarcinoma of the uterus that developed 19 years after her final pregnancy and 4 years after menopause. A total abdominal hysterectomy and bilateral salpingo-oophorectomy was performed. Histopathological examination showed choriocarcinoma of the uterus without components of other germ cell tumors. Karyotype analysis of the tumor cells demonstrated XX. We confirmed its nongestational origin by DNA polymorphism analysis at 15 short tandem repeat loci. After surgery, the patient was given four courses of combination chemotherapy. She is still alive and there has been no evidence of recurrence 3 years after surgery.

Simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry.

In forensic analysis, the identification of urine or human urine among unknown liquids plays an important role. Urea, uric acid, and creatinine are major organic compounds found in human urine. Previous studies have reported that the concentration quotients of these three compounds can be used as an index for the identification of human urine. Here we describe a method for the simultaneous quantification of urea, uric acid, and creatinine in human urine by liquid chromatography/mass spectrometry (LC/MS), with the aim of forensic identification of human urine. Separation of the three analytes was achieved by hydrophilic interaction chromatography, using a TSK gel Amide-80 column with a mobile phase composed of acetonitrile and ammonium acetate aqueous solution, coupled with detection using a mass spectrometer. For quantification, melamine and violuric acid were used as internal standards. Human urine samples were pretreated for LC/MS analysis by dilution with LC mobile phase, followed by centrifugation and filtration. The analytes and internal standards were separated within 9 min. The linear ranges were 2.0-40.0, 0.10-1.60, and 0.13-2.00 mg/mL for urea, uric acid, and creatinine, respectively, with correlation coefficients > 0.99. The intra- and inter-day accuracies of the analytes were - 10.6% to 7.4%, and the precision was within 7.6%. For all analytes, no significant matrix effects were observed and recoveries ranged from 95.4% to 104.6%. Quantitative results of 3 analytes were obtained within their linear range from 10 human urine samples and the quotients, UA/UN × 20 and UA/Cre, were calculated based on previous reports.

A human tissue-based assay identifies a novel carrion blowfly strain for maggot debridement therapy.

Maggot debridement therapy (MDT) is a form of therapeutic wound treatment in which live fly larvae are used intentionally to debride necrotic tissues. MDT has been widely used to treat chronic wounds in humans or animals, such as diabetic foot ulcers. Larvae of a carrion blowfly, Lucilia sericata (green bottle fly), debride wounds by consuming necrotic tissue and removing pathogenic bacteria, promoting effective wound healing. Most medical L. sericata strains were initially collected from natural environments using animal meat as bait and reared on artificial protein-rich media or ground meat. It remains to be examined which strain would be more appropriate for MDT, whereas any method for evaluating the fly's therapeutic potential in humans has not been available. A feeding assay was developed using minced human tissues obtained from surgical waste. To establish L. sericata strains highly eligible for MDT, carrion fly larvae were collected from 45 corpses subjected to forensic autopsy (such as decomposed bodies). Four corpse-derived L. sericata strains were obtained and evaluated using the feeding assay. One strain showed that its feeding activity was 1.4 times higher than the control strain used in conventional MDT. The body length of the adult fly of the corpse-derived strain was longer than the control, which was consistent with the observation that its cell size was enlarged. The human tissue-based assay developed in this study accurately evaluated the ability of fly larvae to debride necrotic wounds. The L. sericata strain newly established from human corpses harboring high feeding activity may offer a clinically significant improvement in MDT.