A practical workflow for forensic species identification using direct sequencing of real-time PCR products.

BACKGROUND: Forensic scientists are often required to identify species of unknown biological samples. Although methods based on sequencing of DNA barcode regions are the gold standard for species identification in single-source forensic samples, they are cumbersome to implement as routine work in forensic laboratories that perform many tests, including human DNA typing. We have developed a species identification workflow that incorporates direct sequencing with real-time PCR products (real-time PCR-direct sequencing) as the technical trick for easy testing in forensic practice. METHOD AND RESULTS: Following our workflow, DNA samples from vertebrates, such as mammals, amphibians, reptiles, birds, and fish, were subjected to species identification using vertebrate universal primers targeting each of the four DNA barcode regions. In real-time PCR melting curve analysis, humans and animals (nonhuman) could be differentiated by comparing melting temperatures, and subsequent real-time PCR-direct sequencing contributed to simplified sequencing. Searches against public DNA databases using the obtained sequences were compatible with the origin of the samples, indicating that this method might be used to identify animal species at the genus level. Furthermore, this workflow was effective in actual casework, which provided rapid test results according to the needs of the investigating agencies. CONCLUSIONS: The species identification workflow will simply sequence as much as possible and can be integrated into routine forensic practice. The real-time PCR-direct sequencing used in this workflow might be beneficial not only for species identification but also for DNA sequencing by using the Sanger method for a variety of life sciences.

An Autopsy Case Report of Homicide by Methanol Intoxication With Pinkish Bilateral Putamina.

ABSTRACT: Many deaths caused by methanol occur as a result of intentional suicide attempts or accidental ingestion, and several investigators have quantified methanol and formic acid in blood and organs. However, to the best of our knowledge, no reports have described regional differences in the concentration of methanol in the brain. A man in his 50s drank alcohol that had been deliberately contaminated with methanol by his wife, and he died of multiple-organ failure after 4 days of intensive medical treatment including hemodialysis. On medicolegal autopsy, cross sections of the brain showed scattered petechial hemorrhage in the brain stem and microscopic hemorrhage with congestion in the bilateral putamina, which showed pinkish discoloration. The concentrations of methanol, formic acid, and ethanol in autopsy samples were measured by headspace gas chromatography, revealing relatively high concentrations of residual methanol and formic acid in the brain (especially in the basal ganglia), although methanol had been eliminated from the blood. Even after 4 days of medical treatment, postmortem toxicological analysis of the brain tissue indicated methanol ingestion. The accumulation of formic acid and the consequent local metabolic acidosis may cause brain lesions.

Blunt traumatic aortic dissection death by falling: an autopsy case report.

A man in his early 60 s who worked at a waste disposal plant had fallen into the refuse pit and was immediately taken to the emergency department for treatment. After 8 days without recovering consciousness, the man died. Antemortem contrast-enhanced computed tomography at the emergency department indicated Stanford type B/DeBakey type IIIb aortic dissection. The autopsy showed a sharp and transverse intimal tear 0.6 cm in length in the aortic isthmus and fractures in the 5th-6th thoracic vertebrae. No structural abnormalities in arterial walls were noted on histopathological examination. The traumatic aortic dissection induced by falling is rare, compared with vehicle crash. Although the verification process was challenging, the cause of death was ultimately concluded as traumatic aortic dissection due to falling into the refuse pit. The following observations were cited as evidence: (1) the location and feature of the intimal tear, (2) the positional relationship between the impact site and the entry tear, and (3) the circumstance of clash impact onto the "cushion" of accumulated waste in the refuse pit. Inquiries into the cause of death, such as those made in this report, are required to provide detailed information on the circumstances of the accident, postmortem examinations, and careful consideration.

Anterior Tricuspid Leaflet Cleft in an Adult Male: An Autopsy Case Report.

The deceased was a 44-year-old male who was treated for a suspected Ebstein's anomaly observed using transthoracic echocardiogram. He was found dead in his bed at home. Autopsy revealed that the septal tricuspid leaflet was intact; however, a large anterior tricuspid leaflet cleft and right atrioventricular cavity dilation were observed. Pathological examination revealed a normal tricuspid valve, except for the presence of a cleft with local fibrosis of the left ventricle papillary muscle and hemosiderin-containing macrophages at both lungs. There were no other abnormalities that may have led to death. It was concluded that he died a cardiac death based on the right heart overload associated with the anterior tricuspid leaflet cleft. This case indicates the possibility that the anterior tricuspid leaflet cleft can cause death and also highlights the necessity of a detailed autopsy to accurately diagnose the cause of death.

Rapid identification of Gloriosa superba and Colchicum autumnale by melting curve analysis: application to a suicide case involving massive ingestion of G. superba.

The plant species Gloriosa superba and Colchicum autumnale produce extremely poisonous colchicine as a major toxic metabolite. Almost all previous studies on colchicine poisoning have focused on drug analysis and clinical and pathological aspects. In this study, we developed a rapid, highly sensitive method to identify G. superba and C. autumnale. This method, which can distinguish between G. superba and C. autumnale using even minute amounts of plant material, is based on duplex real-time PCR in combination with melting curve analysis. To discriminate between the two genera of colchicine-containing plants, we designed new primer pairs targeting the region of the ycf15 gene, which is present in C. autumnale but not G. superba. By producing PCR amplicons with easily distinguishable melting temperatures, we were able to rapidly and accurately distinguish G. superba from C. autumnale. The new primer pairs generated no PCR amplicons from commercially available human DNA or various plant DNAs except for G. superba and C. autumnale. Sensitivity testing indicated that this assay can accurately detect less than 0.031 ng of DNA. Using our method in conjunction with colchicine drug analysis, we successfully identified G. superba in the stomach contents of a suicide victim who ingested massive quantities of a colchicine-containing plant. According to these results, duplex real-time PCR analysis is very appropriate for testing forensic samples, such as stomach contents harboring a variety of vegetables, and enables discrimination between G. superba and C. autumnale in forensic and emergency medical fields.

A simple and versatile authenticity assay of coffee products by single-nucleotide polymorphism genotyping.

Interspecific single-nucleotide polymorphisms (SNPs) in the rbcL DNA barcode have been strictly validated and adopted as a designed SNP genotyping maker to discriminate between two major coffee species, Coffea arabica and C. canephora, and to estimate the mixing ratio of DNA from C. arabica/C. canephora in this study. The SNP genotyping is applicable to not only green (unroasted) coffee beans, but also processed coffee products (roasted coffee beans and instant coffee powder), in which genomic DNA is degraded, because the genotyping developed in this study requires only 10 copies of 63-bp-long DNA fragments of rbcL gene. The authenticity assay established in this study has several advantages: a high versatility to DNA sample conditions; simple and rapid procedures (only two steps; DNA extraction and SNP genotyping); the feasibility in coffee business for practical use to prevent false advertising and provide quality control. Abbreviations: SNP: single-nucleotide polymorphism; SBS: single base substitution; ISR: intergenic spacer region; INDEL: insertion-deletion.

Autopsy Case of a Penetrating Wound to the Left Cerebral Hemisphere Caused by an Accidental Shooting With a Crossbow.

A crossbow is a bow that shoots an arrow when a gun-like trigger is pulled. Deaths caused by accidental crossbow shootings are extremely rare. Here we describe an autopsy case of a penetrating wound to the left cerebral hemisphere caused by an accidental shooting with a crossbow. A man in his early 60s who lived with his wife and had used crossbows for 20 years as his hobby was found one early morning in the shed of his house, collapsed and bleeding from the head and neck. He was taken to a hospital and died after approximately 3 days of conservative treatment. At autopsy, a penetrating wound between the upper part of the left anterior neck and the left frontoparietal region was evident. Traumatic intracerebral hematoma was observed in the left frontal lobe, and severe traumatic subarachnoid hemorrhage was present throughout the brain. Cerebral contusion and hematoma without any organization were noted around the penetration. The cause of death was determined to be cerebral contusion and intracerebral hematoma due to the penetrating wound by the crossbow arrow. He was probably trying to load an arrow into the crossbow by placing it on the floor, pointing upward, and made a mistake in its operation that resulted in the shooting of the arrow. This case is unique because it was a rare accidental death caused by a crossbow arrow, and a detailed histopathological examination was performed.

Postmortem interval estimation: a novel approach utilizing gas chromatography/mass spectrometry-based biochemical profiling.

While the molecular mechanisms underlying postmortem change have been exhaustively investigated, the establishment of an objective and reliable means for estimating postmortem interval (PMI) remains an elusive feat. In the present study, we exploit low molecular weight metabolites to estimate postmortem interval in mice. After sacrifice, serum and muscle samples were procured from C57BL/6J mice (n = 52) at seven predetermined postmortem intervals (0, 1, 3, 6, 12, 24, and 48 h). After extraction and isolation, low molecular weight metabolites were measured via gas chromatography/mass spectrometry (GC/MS) and examined via semi-quantification studies. Then, PMI prediction models were generated for each of the 175 and 163 metabolites identified in muscle and serum, respectively, using a non-linear least squares curve fitting program. A PMI estimation panel for muscle and serum was then erected which consisted of 17 (9.7%) and 14 (8.5%) of the best PMI biomarkers identified in muscle and serum profiles demonstrating statistically significant correlations between metabolite quantity and PMI. Using a single-blinded assessment, we carried out validation studies on the PMI estimation panels. Mean ± standard deviation for accuracy of muscle and serum PMI prediction panels was -0.27 ± 2.88 and -0.89 ± 2.31 h, respectively. Ultimately, these studies elucidate the utility of metabolomic profiling in PMI estimation and pave the path toward biochemical profiling studies involving human samples.

Estimates of exposure to cold before death from immunohistochemical expression patterns of HSP70 in glomerular podocytes.

Environmental factors such as outside temperature at the time of death are very important for forensic diagnoses and police investigations. In particular, death in a cold environment is associated with factors of forensic interest, including hypothermia, drowning in cold water, or postmortem body movement by a suspect. Hypothermia raises a special problem because of the difficulty of evaluation during autopsy. We describe here a unique method of estimating antemortem environmental temperature, involving the immunohistochemical analysis of HSP70 expression patterns in glomerular podocytes. Using this method, we found that HSP70 was present in glomerular podocytes at autopsy and that HSP70 was highly expressed, mainly in the nucleus of podocytes, in deaths associated with exposure to cold. Interestingly, this expression pattern was specific to death in a cold environment, including hypothermia and drowning in cold water. Analysis of the pattern of HSP70 expression in glomeruli may therefore be very useful in forensic diagnosis, for determining whether the antemortem environmental temperature was low. Moreover, immunohistochemical and real-time PCR assays of the molecular mechanism of HSP70 and HSF1 expression in glomeruli following exposure to cold indicated that HSP70 was rapidly translocated to the nucleus of podocytes following exposure to cold, but without new protein synthesis.

Forensic toxicological implications of pleural effusion; an autopsy case of drug overdose.

UNLABELLED: A fatal poisoning case involving multiple psychotropic drugs is presented. Quantitative toxicological analysis showed that the concentrations of phenobarbital, promethazine, chlorpromazine, alprazolam and bromazepam in the femoral blood were 69.51µg/ml, 32.73µg/ml 24.76µg/ml, 0.04µg/ml and 0.31µg/ml respectively, and large amounts of drugs were also detected in the stomach contents. We concluded that the cause of death was mainly due to overdose of phenobarbital, promethazine and chlorpromazine by massive ingestion, and we also discuss the value of pleural effusions as an alternative specimen for toxicological examination. KEYWORDS: multiple drug - poisoning - liquid chromatography mass spectrometry - phenobarbital - pleural effusion.

An autopsy case report of aortic dissection complicated with histiolymphocytic pericarditis and aortic inflammation after mRNA COVID-19 vaccination.

A male in his 90 s consulted a doctor because he experienced several days of general fatigue and dyspnea. He was diagnosed with heart failure, and diuretic medications taken for 3 days relieved his symptoms. However, he was found dead on the morning of the fourth day after consultation. He had received a third dose of coronavirus disease 2019 (COVID-19) vaccine approximately 2 weeks before death. An autopsy revealed dissection of the ascending aorta and pericardial hemotamponade. The heart showed a white villous surface, and the pericardium was fibrously thick. Microscopic examination revealed pericarditis with predominantly macrophage and lymphocyte infiltration. These histological findings were compatible with those of post-vaccination myocarditis. To the best of our knowledge, histopathologically proven pericarditis after COVID-19 vaccination has not been reported. In the present case, extended inflammation of the aortic adventitia was a possible cause of aortic wall fragility followed by dissection.

Immunohistochemical analysis of CD31 expression in myocardial tissues from autopsies of patients with ischemic heart disease.

CD31, a transmembrane protein expressed on endothelial and hematopoietic cells, plays important roles in leukocyte trafficking, mechanotransduction, angiogenesis, vascular permeability, and regulation of cellular responsiveness. CD31 immunoreactivity is employed as a sensitive and specific endothelial marker in diagnostic pathology. In this study, CD31 expression in myocardial tissues from deceased patients with ischemic heart disease and a mouse model of acute myocardial infarction were examined by immunohistochemical staining. We examined 24 neutral formalin-fixed, paraffin-embedded myocardial tissue samples obtained within 48 h postmortem from the autopsies of patients who were diagnosed with ischemic heart disease. CD31 expression was observed in vascular endothelial and endocardial cells. In necrotic myocardium, diffusion of CD31 antigen was observed. Elevated CD31 expression was observed around myocardial cells undergoing remodeling, suggesting that endothelial proliferation occurred at these sites. In contrast, fibrotic myocardial foci did not show upregulated CD31 expression. The same CD31 expression characteristics as those observed in the human samples were observed in the mouse model. CD31 immunostaining as an endothelial and microvasculature marker may be a useful complement to conventional staining techniques currently used in the diagnosis of ischemic heart disease, and may allow the timing and process of myocardial remodeling to be analyzed in detail.

Immunohistochemical analysis of vimentin expression in myocardial tissue from autopsy cases of ischemic heart disease.

Vimentin is a type III intermediate filament cytoskeletal protein that is expressed mainly in cells of mesenchymal origin and is involved in a plethora of cellular functions. In this study, myocardial tissues from patients with ischemic heart disease and a mouse model of acute myocardial infarction were subjected to immunohistochemistry for vimentin. We first examined 26 neutral formalin-fixed, paraffin-embedded myocardial tissue samples from autopsies of patients that were diagnosed with ischemic heart disease within 48 h postmortem. Myocardial cells were negative for vimentin, whereas non-myocardial cells, including vascular endothelium, vascular smooth muscle, fibroblasts, nerve fibers, adipocytes and mesothelial cells, showed positivity. Elevated vimentin expression was observed around myocardial cells undergoing remodeling, suggesting fibroblastic and endothelial proliferation in these locations. By contrast, myocardial foci that were completely fibrotic did not show upregulated vimentin expression. Inflammatory foci including macrophages and neutrophils were clearly visualized with vimentin immunostaining. The same vimentin expression phenomena as those found in human samples were observed in the mouse model. Our study indicates that immunostaining of vimentin as a marker for myocardial remodeling and the dynamics of all non-myocardial cell types may be useful for supplementing conventional staining techniques currently used in the diagnosis of ischemic heart disease.

Quantitative analysis of thiamylal and its metabolite secobarbital using liquid chromatography-tandem mass spectrometry in adipose tissue, serum, and liver.

Thiamylal is an ultrashort-acting barbiturate used for intravenous administration or general anesthesia induction. However, some cases of poisoning and suicide with thiamylal administration have been reported. Additionally, there are few reports on its analysis in the organs and adipose tissue, which requires purification by column chromatography and evaporation. A rapid and sensitive method was developed for quantifying thiamylal and its metabolite, secobarbital, in the adipose tissue, serum, and liver using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were prepared using modified QuEChERS extraction. For adipose tissue samples, an acetonitrile-hexane partitioning step was added to the extraction. This method was applied to investigate a suspected self-poisoning autopsy case. The quantitation accuracy for thiamylal added to porcine pericardial fat (0.18 µg/g), human serum (0.015 µg/mL), and porcine liver (0.18 µg/g) was 103%, 113%, and 95.3%, respectively. The quantitation limits calculated for porcine pericardial fat, human serum, and porcine liver at a signal-to-noise ratio of 10 were 0.06 µg/g, 0.005 µg/mL, and 0.06 µg/g, respectively. In addition, the thiamylal and secobarbital levels in the forensic autopsy case were 140 and 1.5 µg/g, respectively, in myocardial fat; 3.5-4.9 and 0.12-0.20 µg/mL, respectively, in serum; and 6.2-42 and 0.58-1.1 µg/g, respectively, in liver tissue. Thiamylal is especially distributed in the adipose tissue. The thiamylal-to-fat ratio may help estimate the time from administration to death. The developed modified QuEChERS extraction method with acetonitrile-hexane partitioning is suitable for analyzing hydrophobic compounds, such as thiamylal, in the adipose tissue.

Immunohistochemical analysis of von Willebrand factor expression in myocardial tissues from autopsies of patients with ischemic heart disease.

von Willebrand factor (VWF) plays a crucial role in hemostasis and thrombosis. VWF is involved in platelet attachment to the subendothelium, serving as a carrier protein for coagulation factor VIII. In this study, myocardial tissues from deceased patients with ischemic heart disease and a mouse model of acute myocardial infarction were subjected to immunohistochemistry to determine VWF expression. We examined 28 neutral formalin-fixed, paraffin-embedded myocardial tissue samples obtained from the autopsies of patients who were diagnosed with ischemic heart disease within 48 h postmortem. Most myocardial cells were negative for VWF, although some cells showed nonspecific positivity. Elevated VWF expression was observed around myocardial cells undergoing remodeling, suggesting that endothelial proliferation occurred at these sites. In contrast, completely fibrotic myocardial foci did not show upregulated VWF expression. Positivity in fibrin deposition and hemorrhagic sites was observed. The same VWF expression characteristics as those observed in the human samples were observed in the mouse model. VWF immunostaining as an endothelial marker may be a useful supplementation to conventional staining techniques that are currently used in the diagnosis of ischemic heart disease in terms of examining the timing of myocardial remodeling in detail and highlighting the remodeling process.

Determining falling patterns by estimation of horizontal distance and height.

Whether by accident or foul play virtually thousands of fall-related fatalities occur each year. While a number of past studies addressed the relationship between falls, injury, and death, only a small fraction sought to establish an objective index geared specifically towards determining the cause of a particular fall. The primary objective in the present study was to determine the range of attainable horizontal distances in various forms of active and passive falling patterns. The secondary objective involved the capturing of physical motions at the point of impact via 3D motion analyses in order to identify the defining physical characteristics of a particular form of fall. The introduction of live test subjects to these series of experiments added the advent of fear and other psychological factors to the study which are crucial in simulating real-life cases. To corroborate this point, five subjects (three male and two female) expressed their wishes to withdraw from the study, attributing their decision to feeling an inherent danger and fear of the physical aspects of the present study. The ten subjects were made to fall from a height of 3.65 m under 13 conditions of various natures. Footage of the subjects falling was captured on two high-speed video cameras which markedly improved the calculation of 3D coordinates along the subjects' flight path. After extensive calculations, we were successful in determining the maximum attainable horizontal distances in passive falling patterns. Additionally, we found that force applied to the abdominal area results in shorter horizontal distances in comparison to falls where force is directly applied to the posterior side of the body.

Application of fragment analysis based on methylation status mobility difference to identify vaginal secretions.

Identifying vaginal secretions attaching or adhering to a suspect's belongings would be beneficial for reconstructing the events that have taken place during a sexual assault. The present study describes a novel approach to identify vaginal secretions by fragment analysis using capillary electrophoresis, based on the mobility differences of PCR amplicons from bisulfite-treated DNA depending on methylation status. We targeted three genome regions including each of three vaginal secretion-specific methylated CpG sites reported previously: cg25416153, cg09765089, and cg14991487. In all three genome regions, the amplicon peaks for methylated genomic DNA (gDNA) sequences were only detected in vaginal samples, whereas samples of other body fluids (blood, saliva, semen, and deposit on skin surface) only showed amplicon peaks for unmethylated gDNA sequences. In vaginal secretions, the methylation ratio of each of the three targeted regions between samples was variable, while the ratios at the three regions in each sample were similar. Furthermore, commercial vaginal epithelial cells were completely methylated at the three regions. Therefore, vaginal secretion-specific methylation may derive from vaginal epithelial cells present in the sample. In forensic cases with a limited amount of DNA, the reproducibility of a detected peak using the present method is not high due to degradation of DNA by bisulfite treatment and subsequent stochastic PCR bias. However, it was possible to detect peaks from methylated DNA sequences by performing PCR and capillary electrophoresis in triplicate after bisulfite treatment, even when bisulfite treatment was performed using 0.5 ng of gDNA from vaginal secretions. In addition, the level of methylation at each targeted region was found to be stable in vaginal secretions stored for 1 year at room temperature. Therefore, we conclude that detection of the visual peak from vaginal secretion-specific methylated DNA sequence is useful to prove the presence of vaginal secretions. This approach has the potential to analyze multiple marker regions simultaneously, and may provide a new multiplex assay to identify various body fluids.

Immunohistochemical analysis of thrombomodulin expression in myocardial tissue from autopsy cases of ischemic heart disease.

Thrombomodulin is a transmembrane glycoprotein that is ubiquitously expressed on the surface of vascular endothelial cells. Thrombomodulin exerts its anticoagulant effects by combining with thrombin, activating protein C, and inactivating the coagulation factors FVa and FVIIIa. Clinically, thrombomodulin is also known as a marker of vascular injury because it circulates freely in response to endothelial injury. In this study, myocardial tissue from cases of ischemic heart disease was subjected to immunohistochemistry by thrombomodulin. We examined 40 neutral-formalin-fixed, paraffin-embedded myocardial tissue samples from autopsy cases that were diagnosed with ischemic heart disease (within 48 h postmortem). Thrombomodulin expression was observed in vascular endothelial cells between myocardial cells and in mesothelial cells of the epicardium. In necrotic myocardium, diffusion of thrombomodulin, which reflected endothelial injury, was observed. Upregulated thrombomodulin expression was observed around myocardial cells under ongoing remodeling, which suggested endothelial proliferation in these locations. Completed fibrotic foci of the myocardium did not show upregulated thrombomodulin expression. In a mouse model of acute myocardial infarction, the same phenomena as that found in human samples were observed by immunohistochemistry of thrombomodulin. Immunostaining of thrombomodulin, as a marker for endothelial injury or myocardial remodeling, may be useful for supplementing conventional staining techniques in the diagnosis of ischemic heart disease in forensic pathology.

Autopsy case of Rosai-Dorfman disease presenting as fibrinous pericarditis.

Rosai-Dorfman disease (RDD) is a rare non-Langerhans cell histiocytosis that is characterized histopathologically by accumulation of CD68-positive, S100-positive, and CD1a-negative histiocytes. Cardiac involvement of RDD is rare. We report here an autopsy case of cardiac involvement of RDD presenting as fibrinous pericarditis. A 14-year-old Japanese boy complained of loss of appetite and breathing difficulty when lying down. He was found dead on his back in his bedroom. One year before his death, he was diagnosed with RDD after skin biopsy. At autopsy, the deceased was 153 cm in height and weighed 38 kg with systemic edema. He had flat pigmented light-brown spots, as well as many pale reddish-brown papules on the abdomen and both thighs. Cervical and mediastinal lymphadenopathy was observed. A large amount of pleural and ascitic fluid was observed. The spleen weighed 381.9 g and showed splenomegaly. The heart weighed 620 g and showed acute fibrinous pericarditis with adhesion. Abundant fibrin was observed on the epicardial surface. The infiltrating cells were CD68-positive, S100-positive, and CD1a-negative histiocytes. The skin and spleen showed histiocytic involvement. Systemic edema, large amounts of pleural and ascitic fluid, a high brain natriuretic peptide level in blood, and hemosiderin-laden macrophages in the lungs suggested chronic heart failure. We speculate that the cause of death was extranodal cardiac involvement of RDD with chronic heart failure. This case highlights the need for forensic pathologists to perform a complete autopsy to determine the cause of sudden death when cardiac involvement of RDD is present.

Coronary artery stenosis-related perfusion ratio using dynamic computed tomography myocardial perfusion imaging: a pilot for identification of hemodynamically significant coronary artery disease.

The purpose of this study was to evaluate the feasibility of the stenosis-related quantitative perfusion ratio (QPR) for detecting hemodynamically significant coronary artery disease (CAD). Twenty-seven patients were retrospectively enrolled. All patients underwent dynamic myocardial computed tomography perfusion (CTP) and coronary computed tomography angiography (CTA) before invasive coronary angiography (ICA) measuring the fractional flow reserve (FFR). Coronary lesions with FFR ≤ 0.8 were defined as hemodynamically significant CAD. The myocardial blood flow (MBF) was calculated using dynamic CTP data, and CT-QPR was calculated as the CT-MBF relative to the reference CT-MBF. The stenosis-related CT-MBF and QPR were calculated using Voronoi diagram-based myocardial segmentation from coronary CTA data. The relationships between FFR and stenosis-related CT-MBF or QPR and the diagnostic performance of the stenosis-related CT-MBF and QPR were evaluated. Of 81 vessels, FFR was measured in 39 vessels, and 20 vessels (51%) in 15 patients were diagnosed as hemodynamically significant CAD. The stenosis-related CT-QPR showed better correlation (r = 0.70, p < 0.05) than CT-MBF (r = 0.56, p < 0.05). Sensitivity and specificity for detecting hemodynamically significant CAD were 95% and 58% for CT-MBF, and 95% and 90% for CT-QPR, respectively. The area under the receiver operating characteristic curve for the CT-QPR was significantly higher than that for the CT-MBF (0.94 vs. 0.79; p < 0.05). The stenosis-related CT-QPR derived from dynamic myocardial CTP and coronary CTA showed a better correlation with FFR and a higher diagnostic performance for detecting hemodynamically significant CAD than the stenosis-related CT-MBF.