Estimating bloodstain age in the short term based on DNA fragment length using nanopore sequencer.

We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R2 value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.

Development of an Okinawa panel for biogeographic inference of Okinawans.

BACKGROUND: The Precision ID Ancestry Panel with 165 SNP markers was unable to differentiate between mainland Japanese and Okinawa Japanese or to distinguish either of them from other East Asian populations. AIM: An Okinawa panel was developed with the aim of further separating Okinawa Japanese individuals from mainland Japanese and other Asian groups. Seventy-five SNPs were selected using the most informative markers from the literature. Further, 22 SNPs were selected to separate Okinawa Japanese at minimum SNPs. SUBJECTS AND METHODS: Samples were collected from 48 unrelated individuals from mainland Japan and 46 unrelated residents of the Okinawa prefecture. Data were evaluated by STRUCTURE, principal component, and GenoGeographer analyses. RESULTS: The 22 SNP set had similar levels of differentiation in STRUCTURE and PCA analyses as the 75 SNP set. GenoGeographer analysis showed that, out of the 46 Okinawa Japanese individuals, the 75 SNP and 22 SNP sets correctly assigned the Okinawan population as the most likely population of origin for 32 and 31 individuals, respectively. CONCLUSION: Neither SNP set could completely differentiate between Okinawa Japanese and other Asian groups, however, these sets should be useful for crime investigation, when the sample, cost and time are limited.

Effect existence of aging on stutter ratio evaluated via Bayesian inference.

The stochastic behavior of the stutter ratio (SR) in capillary electrophoresis-based DNA typing is currently described and predicted using statistical models in forensic genetics. Clarifying this behavior can help obtain more objective and robust evidence to the court in terms of mixture interpretation. This study aimed to investigate the effect existence of aging on SR via a Bayesian framework. Nail scrapings and clippings were collected from 68 healthy individuals with informed consent. Samples were classified by age-class: young group (0-16 years; n = 36) and older-adult group (>61 years; n = 32). Then, they were compared in terms of their SRs for each simple repeat locus included in GlobalFiler Kit. Bayesian modeling was performed with lognormal distribution model, which implemented multiple linear regression, allele and age-class as explanatory variables. For all simple repeat loci, the median of the posterior distribution of the age-class parameter was a positive value. For CSF1PO and D7S820, the 95% credible interval of the posterior distribution did not include 0. Our data suggested that aging slightly increases the SR. These findings might help elucidate the stochastic behavior of SR.

Simplified detection of the species of origin of antler velvets using single-stranded tag hybridization chromatographic printed-array strip.

In this study, we developed a convenient and easy-to-use origin identification method for antler velvets based on a simple DNA extraction technique and single-stranded tag hybridization chromatographic printed-array strip (STH-PAS). The primer sets used to detect Cervus elaphus, Rangifer tarandus, and 12S rRNA did not engage in non-specific reactions such as primer dimer formation. In both the triplex and singleplex assays, the sensitivity was < 1 ng DNA. Moreover, Cervus elaphus DNA could be detected in OTC crude drug products. Although the detection sensitivity resulting from the simplified extraction was slightly lower than that obtained with extraction by conventional methods, the amount of DNA was sufficient even from a small sample. The choice of a triplex or singleplex assay will depend on the purpose of the test. For example, if it is important to determine whether the antler velvet is derived from Cervus elaphus or Rangifer tarandus, a triplex assay is appropriate. If it is necessary to explore whether antler velvet from Cervus elaphus is included in an OTC crude drug product, a singleplex assay using the Cervus elaphus primer set is informative. If it is necessary to explore whether powdered antler velvet includes counterfeit products (from Rangifer tarandus), a singleplex assay employing the Rangifer tarandus primer is appropriate. The singleplex assay detects minor components even at a 1,000:1 ratio. Our study thus demonstrated the utility of a method combining simple DNA extraction with STH-PAS for efficient identification of the origin of antler velvets.

Novel parasitic chytrids infecting snow algae in an alpine snow ecosystem in Japan.

Introduction: Microbial communities are important components of glacier and snowpack ecosystems that influence biogeochemical cycles and snow/ice melt. Recent environmental DNA surveys have revealed that chytrids dominate the fungal communities in polar and alpine snowpacks. These could be parasitic chytrids that infect snow algae as observed microscopically. However, the diversity and phylogenetic position of parasitic chytrids has not been identified due to difficulties in establishing their culture and subsequent DNA sequencing. In this study, we aimed to identify the phylogenetic positions of chytrids infecting the snow algae, Chloromonas spp., bloomed on snowpacks in Japan. Methods: By linking a microscopically picked single fungal sporangium on a snow algal cell to a subsequent sequence of ribosomal marker genes, we identified three novel lineages with distinct morphologies. Results: All the three lineages belonged to Mesochytriales, located within "Snow Clade 1", a novel clade consisting of uncultured chytrids from snow-covered environments worldwide. Additionally, putative resting spores of chytrids attached to snow algal cells were observed. Discussion: This suggests that chytrids may survive as resting stage in soil after snowmelt. Our study highlights the potential importance of parasitic chytrids that infect snow algal communities.

A method for determining the origin of crude drugs derived from animals using MinION, a compact next-generation sequencer.

We evaluated whether MinION, an inexpensive portable sequencer, can be used to identify the origin of crude drugs derived from animals. Standard and nonstandard crude drugs with different species of origin were examined. In addition, standards mixed with nonstandard samples were used. As a target gene, cytochrome c oxidase I was amplified and sequenced. The fast mode results had a slightly lower match ratio than high-accuracy mode, but the animals of origin were correctly determined by BLAST for all samples. For antler velvet derived from Rangifer tarandus, even when the sequences were aligned based on Cervus elaphus, the animal of origin was determined correctly. Minor contents could be detected from mixtures of two animals, if the mixtures contained at least 19:1 mtDNA when the coverage allele-fraction threshold was 0.05. By contrast, in fast mode, two sequences could not be separated due to the low accuracy of the base-calling for each read. For fieldwork, the species of origin of crude drugs could be identified with only simple DNA extraction and library preparation. Therefore, MinION appears to be a convenient tool for identifying the origins of crude drugs derived from animals.

Highly sensitive sex determination method using the exon 1 region of the amelogenin gene.

Sex determination is a crucial factor in the identification of unidentified human remains. Sex determination by DNA analysis is particularly useful because it can be applied to samples for which morphological characteristics are unavailable. Because samples handled in forensic DNA typing are easily degraded by environmental factors and microorganisms, there is a need for a method that can accurately determine sex even in highly decayed samples. Previous studies mainly used sex differences in an intron of the amelogenin gene. However, this region is highly polymorphic, and there are cases where accurate sexing cannot be performed because of genetic mutations in the target region. Thus, for reliable sex determination, it is desirable to select loci with as few non-sexual polymorphisms as possible. In this study, we focused on the exon 1 region of the amelogenin gene, which has very little polymorphism other than sex differences. We developed a primer set for sex determination and compared it with the GlobalFiler™ PCR Amplification Kit (GF), which is widely used for forensic DNA typing. The results showed that the amount of DNA required for accurate sex determination was 25 pg for both methods, achieving equivalent sensitivity. Next, we compared the two methods using ancient human skeletons and found that the present method with its shorter amplicon was considerably superior to GF. The present method is simple, rapid, inexpensive, and suitable for analyzing highly degraded samples. Therefore, this method is expected to contribute to forensic sciences and physical anthropology.

Glyceraldehyde-derived advanced glycation end-products are associated with left ventricular ejection fraction and brain natriuretic peptide in patients with diabetic adverse cardiac remodeling.

Objectives: Glyceraldehyde-derived advanced glycation end-products (Glycer-AGEs) have a strong binding affinity for their cognate receptor and elicit oxidative stress and inflammation. However, it remains unknown whether the levels of Glycer-AGEs correlate with the severity of cardiac function and heart failure in patients with diabetic adverse cardiac remodeling (DbCR). Fourteen heart failure patients with type 2 diabetes mellitus (DM) without other cardiac disorders (DbCR group) were enrolled. Another 14 patients with idiopathic dilated cardiomyopathy (DCM) without DM were served as a control (DCM group). All patients were assessed for serum Glycer-AGEs, nitrotyrosine (NT), and tumor necrosis factor alpha (TNFα) and for plasma brain natriuretic peptide (BNP). The left ventricular ejection fraction (LVEF) was evaluated by echocardiography. Results: The mean serum levels of Glycer-AGEs, NT, and TNFα in the DbCR group were significantly higher than those in the DCM group (for Glycer-AGEs, p = .0073; for NT, p = .005; for TNFα, p < .0001, respectively). In the patients with DbCR, the levels of serum Glycer-AGEs and TNFα were closely associated with LVEF and BNP values. Conclusions: Both Glycer-AGEs and TNFα showed close associations with LVEF and the levels of BNP in patients with DbCR. Glycer-AGEs and TNFα may play a pathological role in the development of DbCR.

Increased plasma glutamate in non-smokers with vasospastic angina pectoris is associated with plasma cystine and antioxidant capacity.

Objectives. Endothelial dysfunction caused by oxidative stress plays an important role in the development of vasospastic angina pectoris (VSAP). Glutamate causes endothelial dysfunction by generating oxidative stress, and it inhibits cystine import into endothelial cells via the cystine/glutamate antiporter (XC-), which leads to depletion of antioxidant glutathione. However, whether glutamate and cystine are implicated in the pathogenesis of VSAP remains unclear. We investigated plasma glutamate and cystine levels, oxidative stress markers and antioxidant capacity in non-smoker patients with VSAP to determine whether glutamate and cystine are associated with the development of VSAP. We assessed 49 non-smokers assigned to groups with (n = 27) and without (n = 22) VSAP, and also measured plasma glutamate, cystine, nitrotyrosine, reactive oxygen metabolites and biological antioxidant potential. Results. Plasma glutamate and cystine values were significantly higher in the group with, than without VSAP (59.8 ± 25.7 vs. 43.5 ± 18.7 µmol/L, p = .016 and 35.3 ± 14.2 vs. 25.2 ± 9.1 µmol/L, p = .0056, respectively). Plasma glutamate and cystine values were significantly and positively associated (r = 0.32, p = .027). Levels of the oxidative stress markers nitrotyrosine and reactive oxygen metabolites, and biological antioxidant potential of as a measure of antioxidant capacity, did not significantly differ between the two groups. However, glutamate and biological antioxidant potential values were significantly and negatively associated (r = -0.3, p = .036). Conclusion. Plasma glutamate levels were increased in patients with VSAP who did not smoke, and they were positively associated with plasma cystine and negatively associated with the biological antioxidant potential levels.

A sudden infant death associated with atrial septal aneurysm and abnormality of the connecting site between the inferior vena cava and right atrium.

An atrial septal aneurysm (ASA) is a rare cardiac anomaly characterized by varicose bulgingofthe atrial septum (oval fossa) into the left or right atrium. Pathogenesis and clinical significance of ASA are controversial. We report an autopsy case of a huge undiagnosed ASA with abnormality of the connecting site between the inferior vena cava and the right atrial ostium in a 2-month-oldJapanesefemale who died suddenly and unexpectedly. She was born at 36 weeks 4 days (body weight 3,110 g). No abnormality was detected during pregnancy or delivery. The postnatal growth was normal with no cardiac problem detected at the 1-month checkup. The ASA bulged off in a mass to the left atrium (width, 0.8 cm; excursion ratio, 53%), reaching close to the inflow site of the right pulmonary vein, with dilation of the pulmonary vein. The connecting site between the inferior vena cava and the right atrium was atypically located 1.6 cm away from the atrioventricular groove. Although most cases of ASA in an infant resolve physiologically as the infant grows, the infant in the present case is thought to have had an exceptional pathological ASA, possibly causing supraventricular arrhythmia. The abnormality of the connecting site between the inferior vena cava and the right atrium might have affected the development and continuation of the ASA.

Diagnostic significance of the histopathology of bone marrow macrophages in forensic autopsies.

Forensic pathologists often encounter autopsies that require an assessment of antemortem general conditions (e.g., infection, metabolic disorders). To establish evaluation clues for such cases, we quantitatively examined macrophages and the general pathology of bone marrow in samples from 180 forensic autopsy cases of decedents with various conditions. Hematoxylin-eosin staining, Berlin blue staining, and immunostainings for CD163, CD138, and CD61 were performed. We determined the numbers per field (density) of total macrophages, swollen macrophages, macrophages with hemophagocytosis, and hemosiderin-laden macrophages. Each density was standardized by identifying its ratio to the total number of macrophages. The decedents' background data (cause of death, other pathological findings, postmortem interval, antemortem symptoms, and presence of resuscitation) were extracted. No correlations were found between the postmortem interval and the other decedent data, indicating that these data are not affected by postmortem changes. In the group in which inflammatory disease was the cause of death, there were significant elevations in the ratio of the swollen macrophage density to total macrophages. Significantly higher ratios of the density of swollen and hemophagocytic macrophages were observed in the group in which conditions with a prolonged agonal period were the cause of death. The group with a return of spontaneous circulation to resuscitation showed a significantly higher ratio of macrophage density with hemophagocytosis. This study provides the first statistical analysis focused on bone marrow histopathology in forensic autopsies. The results will be useful for elucidating causes of death and agonal-period conditions.

Estimating individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq.

We tried to estimate individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq. The BAM files produced by MiSeq were viewed using Integrative Genomics Viewer (IGV) to verify mixed bases. By sorting the reads according to base type for each mixed base, partial haplotypes were determined. Then, the BAM files produced by MinKNOW were viewed using IGV. To determine haplotypes with IGV, only mixed bases determined by MiSeq were used as target bases. By sorting the reads according to base type for each target base, each contributor's haplotype was estimated. In mixed samples from two contributors, even a haplotype with a minor contribution of 5% could be distinguished from the haplotype of the major contributor. In mixed samples of three contributors (mixture ratios of 1:1:1 and 4:2:1), each haplotype could also be distinguished. Sequences of C-stretches were determined very inaccurately in the MinION analysis. Although the analysis method was simple, each haplotype was correctly detected in all mixed samples with two or three contributors in various mixture ratios by combining MinION and MiSeq. This should be useful for identifying contributors to mixed samples.

Removal effect of DNA contamination by hydrogen peroxide plasma compared to ethylene-oxide gas.

We examined the ability of hydrogen peroxide plasma (HPP) to remove DNA contamination, to evaluate whether it is a suitable forensic-grade treatment under ISO 18385. HPP treatment was compared to ethylene-oxide gas (EOG) treatment, which is required by ISO 18385. For the evaluation, commercial control DNA solution and cultured cells sprinkled on Petri dishes were used, and the DNA fragments (214 and 80 bp autosomal DNA fragments and 75 bp Y chromosome fragment) were quantified. HPP treatment was performed up to four times and EOG treatment was performed once. Performing HPP treatment three times was as effective as EOG treatment, with all fragments decreasing to below 1/1,000 in DNA solution. With STR and Y-STR typing, no alleles were detected for three HPP treatments of control DNA using the original amount, i.e., 1 ng. Therefore, HPP appears useful for removing DNA contamination. For cells sprinkled on Petri dishes, the DNA degradation abilities of the HPP and EOG were comparable. However, less DNA was degraded with the HPP and EOG and neither met the ISO criteria. Although the current version of ISO 18385 recommends an evaluation method using cultured cells sprinkled on Petri dishes, it needs to be revised. These findings should be considered when revising ISO 18385.

Estimating included animal species in mixed crude drugs derived from animals using massively parallel sequencing.

We developed a method that can detect each animal species of origin for crude drugs derived from multiple animal species based on massively parallel sequencing analysis of mitochondrial genes. The crude drugs derived from animals investigated in this study were Cervi Parvum Cornu and Trogopterorum feces, which are derived from a mix of different animal species, two chopped cicada sloughs, and two commercial Kampo drugs. The mitochondrial 12S rRNA, 16S rRNA, and cytochrome oxidase subunit I gene regions were amplified and sequenced using MiSeq. The ratios of haplotype to total number of sequences reads were calculated after sequence extraction and trimming. Haplotypes that exceeded the threshold were defined as positive haplotypes, which were compared with all available sequences using BLAST. In the Cervi Parvum Cornu and Trogopterorum feces samples, the haplotype ratios corresponded roughly to the mixture ratios, although there was a slight difference from mixture ratios depending on the gene examined. This method could also roughly estimate the compositions of chopped cicada sloughs and Kampo drugs. This analysis, whereby the sequences of several genes are elucidated, is better for identifying the included animal species. This method should be useful for quality control of crude drugs and Kampo drugs.

Modeling the minus two base pair stutter ratio of the D1S1656 locus: A sequence-based mixture distribution model.

In this study, we propose a stutter ratio for a minus two base pair stutter (-2bpSR) model of the D1S1656 locus in capillary electrophoresis (CE)-based short tandem repeat (STR) typing. DNA from a total of 108 Japanese individuals was analyzed via massively parallel sequencing to investigate the length of the longest uninterrupted stretch of two base repeat motif (2bpLUS value) within repetitive structures involving the flanking region. Additionally, -2bpSR data was collected using the GlobalFiler Kit on a 3500xL Genetic Analyzer. As a result of sequencing analysis, all alleles were classified into two types by their 2bpLUS values. The -2bpSR differed significantly between the types. Then, we modeled the -2bpSR with a mixture log-normal distribution using the classification of alleles based on the 2bpLUS values. Furthermore, probabilities of the sequence type within each repeat number in the mixture log-normal distribution model were estimated using logistic regression for each of the five major detected populations. This study is expected to enable interpretation of STR typing while considering minus two base pair stutter at the D1S1656 locus.

Bloodstain examination and DNA typing from hand-washed bloodstains on clothes.

We investigated whether bloodstain examination and DNA typing can be performed on washed bloodstains on clothes. Blood was dropped onto T-shirts made from 100% cotton or 100% polyester. After drying, the T-shirts were hand-washed with handwashing soap, dishwashing detergent, laundry detergent, soap, or just water until the bloodstains could not be seen. After drying the T-shirts, DNA and RNA were extracted simultaneously from the bloodstained areas using commercial kits. RNA was reverse-transcribed to DNA, and then the detection of the mRNAs for HBB, ACTB, and 18S rRNA was examined. DNA was quantified via real-time PCR, and then STR typing was performed with a commercial kit. The luminol and leucomalachite green tests were used as preliminary bloodstain tests, and an immuno-chromatography kit was used to identify human bloodstains. DNA could be extracted from all washed bloodstains, but more DNA was extracted from cotton T-shirts than from polyester T-shirts. STR typing was successful for all bloodstains without issues such as PCR inhibition. In the human bloodstain identification test using mRNA, almost all bloodstains produced a Ct value for HBB and all bloodstains produced a Ct value for 18S rRNA, whereas few bloodstains produced a Ct value for ACTB. All bloodstains reacted positively to luminol, but some were negative for leucomalachite green. Most of the bloodstains did not react positively in the human bloodstain identification test using the immuno-chromatography kit. The results suggest that human bloodstain identification and DNA typing can still be performed after clothes with bloodstains are washed.

A Noise Tolerant Spread Spectrum Sound-Based Local Positioning System for Operating a Quadcopter in a Greenhouse.

Quadcopters are beginning to play an important role in precision agriculture. In order to localize and operate the quadcopter automatically in complex agricultural settings, such as a greenhouse, a robust positioning system is needed. In previous research, we developed a spread spectrum sound-based local positioning system (SSSLPS) with a 20 mm accuracy within a 30 × 30 m greenhouse area. In this research, a noise tolerant SSSLPS was developed and evaluated. First, the acoustic noise spectrum emitted by the quadcopter was documented, and then the noise tolerance properties of SSSounds were examined and tested. This was done in a greenhouse with a fixed quadcopter (9.75 N thrust) with the positioning system mounted on it. The recorded quadcopter noise had a broadband noise compared to the SSSound. Taking these SSSound properties into account, the noise tolerance of the SSSLPS was improved, achieving a positioning accuracy of 23.2 mm and 31.6 mm accuracy within 12 × 6 m for both Time-division Multiple Access (TDMA) and Frequency-division Multiple Access (FDMA) modulation. The results demonstrate that the SSSLPS is an accurate, robust positioning system that is noise tolerant and can used for quadcopter operation even within a small greenhouse.

The origin identification method for crude drugs derived from arthropods and annelids using molecular biological techniques.

We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots.

Estimation of the number of contributors to mixed samples of DNA by mitochondrial DNA analyses using massively parallel sequencing.

We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209-16,400) and B- (positions 30-284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics.

Cardiac arrest induced by the intentional ingestion of boric acid and mirtazapine treated by percutaneous cardiopulmonary bypass: a case report.

BACKGROUND: Mirtazapine has a good tolerability and safety profile that demonstrates several benefits over other antidepressants and it is associated with few fatalities. Boric acid is an odorless white powder that is generally not recognized as a poisonous substance. We report a case of cardiac arrest induced by the intentional ingestion of mirtazapine, boric acid, and sennosides, by a patient who required percutaneous cardiopulmonary bypass. CASE PRESENTATION: Our patient was a 49-year-old Japanese woman with a history of depression; she was found in an unconscious state after ingesting boric acid (unknown amount), mirtazapine (1950 mg), and sennosides (780 mg). On arrival, she was in a deep coma with marked hypotension induced by atrial fibrillation, tachycardia, and diffuse hypokinetic cardiac motion. She had systemic diffuse erythema. Her serum concentrations of boric acid and mirtazapine on arrival were 560.49 mg/L and 1270 ng/mL, respectively. She experienced repeated cardiac arrest, and was therefore treated with tracheal intubation, mechanical ventilation, percutaneous cardiopulmonary bypass, and continuous hemodialysis filtration. Stable circulation and respiration and a normal kidney function were finally obtained and she was transferred to a local medical facility in a persistent unconscious state. CONCLUSIONS: This is the first case of a return of spontaneous circulation after cardiac arrest induced by the intentional ingestion of boric acid and mirtazapine, requiring percutaneous cardiopulmonary bypass for survival. To maintain cerebral perfusion during percutaneous cardiopulmonary bypass, even in a prolonged state of cardiac arrest induced by overdose, is medically, ethically, and economically challenging.