An Autopsy Case in Which Self‐Bloodletting Via a Cervical Blood Access Led to a Fatal Outcome*

Abstract: A 48‐year‐old woman was found dead on a chair in her living room. She had received dialysis every day because of chronic renal failure for the past 15 years. On a table beside her, there was a mirror and 10‐mL syringe on a napkin. A stopper was out of place in a portion of a three‐way blood access tube established in the right cervical region, and blood coagulation was noted in the lumen. There was a bloodstained measuring cup on the floor. Autopsy findings included a large number of shunt traces in the bilateral infraclavicular fossae and upper limbs, as well as the cervical blood access terminal reaching the right atrium via the internal jugular vein to superior vena cava. Various organs showed anemia. Neither a fatal lesion nor injury was noted in the main organs. Therefore, this patient may have committed suicide by self‐bloodletting via a cervical blood access.     

Assessment of the Role Played by N‐propanol Found in Postmortem Blood in the Discrimination Between Antemortem Consumption and Postmortem Formation of Ethanol Using Rats

This study disproves the reliability of n‐propanol as a biomarker to establish whether the ethanol found in postmortem blood is derived from antemortem ingestion or postmortem putrefactive processes. Two groups of rats were given ethanol or normal saline solution, respectively, and sacrificed 1.5 h later. After putrefaction, blood and, in a few cases, urine samples from the rats were analyzed for ethanol and n‐propanol by head‐space gas chromatography equipped with flame ionization detection. Although the concentration ratios of ethanol/n‐propanol in the postmortem blood collected from the bodies without prior alcohol consumption were expected to be <20 (as per limited case reports and previous in vitro studies), in samples from several rats that were on saline solution, this ratio was found to exceed 20. In conclusion, the concentration ratio of ethanol/n‐propanol in postmortem blood does not allow for the discernment between antemortem ingestion and the postmortem synthesis of ethanol.     

Determination of Nitrite in Whole Blood by High‐Performance Liquid Chromatography with Electrochemical Detection and a Case of Nitrite Poisoning

Although nitrite is widely used in meat processing, it is a major toxicity hazard to children and is responsible for the blue‐baby syndrome. A simple and effective method to determine nitrite in whole blood has been devised using ion chromatography with suppressed conductivity detection. The blood sample was deproteinized by adding acetonitrile and purified with mini‐cartridges to remove hydrophobic compounds, chloride ions, and metal ions. An aliquot of the filtrate was injected onto the ion chromatography. The retention time for nitrite was 13.8 min and the detection limit of nitrite in whole blood was 0.4 μmol/L. The calibration curve was linear (r² = 0.9999) over the concentration working range. The blood nitrite concentration of a victim who attempted suicide by ingesting sodium nitrite powder was determined using the present method. The basal levels for nitrite in human blood was determined with 7.1 ± 0.9 μmol/L (n = 12).     

Determination of Acetamiprid and IM‐1‐2 in PostMortem Human Blood,Liver, Stomach Contents by HPLC‐DAD

An HPLC‐DAD method was developed to detect and quantify a neonicotinoid insecticide acetamiprid (ATP) and its metabolite IM‐1‐2 in autopsy samples of a fatal intoxication case. The postmortem blood and tissue distribution of ATP and IM‐1‐2 was determined for the first time. The method showed acceptable precisions and recoveries with relative standard deviations of <10% for ATP level and 1.38 % for IM‐1‐2. The detection and quantification limits for ATP were 0.015 μg/mL and 0.030 μg/mL for blood and were 0.035 μg/g and 0.050 μg/g for liver samples, respectively. The mean contents of ATP were 0.79 μg/g in the liver, 47.35 μg/g in the stomach contents and 2.7 μg/mL in the blood. IM‐1‐2 content was 17.0 μg/g in the stomach contents. ATP and IM‐1‐2 were not detected in the urine. The presence of ATP and IM‐1‐2 in the samples was confirmed by GC‐MS. The method can be exploited in future forensic casework.     

Distribution of Enantiomers of Methadone and Its Main Metabolite EDDP in Human Tissues and Blood of Postmortem Cases

Knowledge concerning the distribution of methadone in postmortem human tissue and the effect of postmortem redistribution on methadone is today limited making the choice of a suitable substitute for femoral blood difficult when this is not available. Cardiac blood, femoral blood, muscle, and brain tissue concentrations of the enantiomers of methadone and its metabolite 2‐ethyl‐1,5‐dimethyl‐3,3‐diphenylpyrrolinium were recorded for 155 postmortem cases. Brain and muscle tissue concentrations exceeded the femoral blood concentrations with a median fold of 2.3 and 1.6, respectively, but both had a better correlation than cardiac blood to femoral blood concentrations. The Kruskal–Wallis test showed a significant dependency on time and body mass index for some of the matrix ratios over femoral blood. We conclude brain or muscle tissue may constitute a better alternative for measurement of methadone than cardiac blood for situations in which femoral blood is not available, despite concentrations in both matrices being systematically higher.     

Sudden Death from Cardiopulmonary Arrest on Arrival of a Patient with Pulmonary Tuberculosis: A Case Diagnosed by Postmortem CT and Autopsy

Sudden death due to massive hemoptysis during management of tuberculosis occurs in a considerable number of patients. However, when massive airway hemorrhage occurs in a patient in whom tuberculosis has not been confirmed and a blood is not apparent externally on the face/body, it is difficult to immediately identify the cause of death as airway obstruction by tuberculous bleeding in the airway. We encountered an 83‐year‐old Japanese woman with her medical history included treatment of tuberculosis in her 20s who was in cardiopulmonary arrest on arrival (CPAOA), and the cause of sudden death could not initially be identified. Postmortem CT (PMCT) and autopsy revealed that the cause of sudden death was airway obstruction/asphyxia by tuberculous massive airway hemorrhage. Identification of the cause of death facilitated a subsequent active contact investigation and led to prevention of secondary tuberculosis infection.     

Site‐, Technique‐, and Time‐Related Aspects of the Postmortem Redistribution of Diazepam,Methadone, Morphine,and their Metabolites: Interest of Popliteal Vein Blood Sampling

Sampling site, technique, and time influence postmortem drug concentrations. In 57 cases, we studied drug concentration differences as follows: subclavian vein‐dissection/clamping versus blind stick, femoral vein‐dissection/clamping versus blind stick, right cardiac chamber, and popliteal vein‐dissection and clamping only. Cases were distributed in group #1 (all cases with both techniques), group #2 (dissection/clamping), and group #3 (blind stick). Sampled drugs were diazepam, methadone, morphine, and their metabolites. To assess PMR, mean concentrations and ratios were calculated for each group. Time‐dependent variations of blood concentrations and ratios were also assessed. Results indicate that site, method, and time may influence postmortem distribution interpretation in different ways. Popliteal blood seems less subject to PMR. In conclusion, our study is the first to evaluate concurrently three main aspects of PMR and confirms that the popliteal vein may represent a site that is more resistant to the changes seen as a result of PMR.     

Isolation and Individualization of Conceptus and Maternal Tissues from Abortions and Placentas for Parentage Testing in Cases of Rape and Abandoned Newborns*

Abstract: Abortion specimens are often submitted to forensic laboratories as the only piece of physical evidence in rape and incest cases. The recovery of conceptus tissues from this evidence permits the use of paternity testing to evaluate suspects. In cases of abandoned newborns, the recovery of maternal tissue from the placenta allows for the direct comparison of genetic profiles between the suspected mother and the biological mother. We report on the identification and isolation of conceptus tissues from embryonic‐ and fetal‐period abortions, and maternal tissues from delivered placentas, by gross and low‐magnification examination with manual dissection. Hundreds of single‐source samples have been successfully recovered by this method and short tandem repeat typed using standard forensic procedures. We additionally describe extraembryonic tissues that can be recovered and typed in the absence of the embryo proper. We conclude that an expertise and protocols can be developed by forensic laboratories for the routine analysis of this evidence.     

A New Approach for the Carbon Monoxide (CO) Exposure Diagnosis: Measurement of Total CO in Human Blood Versus Carboxyhemoglobin (HbCO)

The aim of the study is to present the application of a headspace–gas chromatography–mass spectrometry (HS‐GC‐MS) method for the determination of the carbon monoxide (CO) blood concentration and to compare it with carboxyhemoglobin (HbCO) saturation. In postmortem cases, the HbCO measured by spectrophotometry frequently leads to inaccurate results due to inadequate samples or analyses. The true role of CO intoxication in the death of a person could be misclassified. The estimation of HbCO from HS‐GC‐MS CO measurements provides helpful information by determining the total CO levels (CO linked to hemoglobin (HbCO) and CO dissociated from hemoglobin). The CO concentrations were converted in HbCO saturation levels to define cutoff blood CO values. CO limits were defined as less than 1 μmol/mL for living persons, less than 1.5 μmol/mL for dead persons without CO exposure, and greater than 3 μmol/mL for dead persons with clear CO poisoning.     

A Rare Case of Poisoning with a Volatile Substance: Quantitative Determination of n‐Butane in Postmortem Tissue

Poisoning with volatile substances remains exceptional. Authors report the case of a married couple who were found in a car with a butane gas bottle: the woman was dead and her husband alleged it was an unsuccessful suicide pact. A specific research of volatile substances on postmortem samples with headspace gas chromatography–mass spectrometry following a quantitative determination was performed. The n‐butane concentrations detected were composed of 610 μg/L (cardiac blood), 50 μg/kg (brain), 134 μg/kg (lungs), 285 μg/kg (liver), and 4090 μg/kg (heart) and were compatible with the rare lethal concentrations evoked in the literature. The cause of death was determined to be asphyxiation through n‐butane criminal poisoning. Authors recommendation therefore is to take samples immediately and place them in properly sealed containers and hence analyzing the samples as soon as possible after collecting them or storing them under ?30°C (?22°F) if analyses cannot be performed immediately.     

Postmortem Blood Concentrations of R‐ and S‐Enantiomers of Methadone and EDDP in Drug Users: Influence of Co‐Medication and P‐glycoprotein Genotype

Abstract: We investigated toxicological and pharmacogenetic factors that could influence methadone toxicity using postmortem samples. R‐ and S‐methadone were measured in femoral blood from 90 postmortem cases, mainly drug users. The R‐enantiomer concentrations significantly exceeded that of the S‐enantiomers (Wilcoxon’s test, p < 0.001). The samples were divided into four groups according to other drugs detected (methadone only, methadone and strong analgesics, methadone and benzodiazepines, or methadone and other drugs). There was no significant difference in any of the R‐methadone/total methadone ratios among the four groups. The median R/S ratio was 1.38, which tends to be higher than that reported for the plasma of living subjects. In addition, we investigated whether small nucleotide polymorphisms in the MDR1 gene that encode the drug transporter P‐glycoprotein were associated with the concentrations of R‐ and S‐methadone and its metabolite 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine. No significant association was detected.     

Simultaneous Determination of 13 Anticoagulant Rodenticidesin Human Blood by Liquid Chromatography–Tandem Mass Spectrometry and its Application in Three Poisoning Cases

Anticoagulant rodenticides are widely used for rodent control around the world. A rapid and sensitive method was developed and validated for the simultaneous determination of 13 anticoagulant rodenticides (coumafuryl, pindone, valone, warfarin, coumatetralyl, coumachlor, diphacinone, dicumarol, chlorophacinone, bromadiolone, difenacoum, flocoumafen, and brodifacoum) in human blood by liquid chromatography–tandem mass spectrometry. After liquid–liquid extraction, the anticoagulant rodenticides were separated on an Eclipse Plus C18 column. Linearities were observed for each analyte in blood ranging from 0.5 to 50 ng/mL, with correlation coefficients over 0.99. The limits of detection ranged from 0.01 to 0.2 ng/mL, and the limits of quantification were 0.5 ng/mL for all analytes. The intraday and interday precisions were <15%, and accuracies ranged from 80.3% to 111.0%. This validated method with high sensitivity has been applied in three anticoagulant rodenticide poisoning cases and has been used successfully in monitoring blood concentrations for months.     

Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples*†

Abstract: Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C‐stretch and CA repeat. To observe which CA “alleles” were present in each tissue, PCR products were cloned and re‐sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.     

Preparation of Artificial Blood from the Extract of Legume Root Nodules,and the Creation of Artificial Latent Fingermarks in Blood Using Artificial Blood,

Distribution of homogeneous fingermarks in blood is essential for conducting proficiency tests in forensic science. Hence, the artificial blood was prepared using the root nodule extract of Glycine max plants. The reactivity of the artificial blood with widely used human blood detection reagents was tested. Artificial latent fingermarks in blood were printed using an inkjet cartridge case filled with artificial blood solution. The artificial latent fingermarks in blood were developed with amino acid‐sensitive reagents and could obtain development as prominent as the image of the master fingermark saved on the computer. Therefore, it has been confirmed that the extract of legume root nodules can be used as artificial blood, and the artificial blood can be used for the preparation of artificial latent fingermarks or footmarks in blood.     

Suicide by Self‐Administration of a Drug Mixture (Propofol,Midazolam, and Zolpidem) in an Anesthesiologist: The First Case Report in Italy

The authors report an unusual case of suicide of an anesthesiologist, in which the suicide manner and means depend upon the victim's occupation. This is the first case report published in Italy of a death involving propofol and other drugs. The anesthesiologist was found dead with an empty drip still inserted in the hand and another one near his body. Forensic and toxicological findings suggested that the cause of death was a respiratory depression due to a self‐administration of a rapidly infused lethal drug mixture. Analytical drug quantification was performed by gas chromatography‐mass spectrometry. Blood analysis revealed: zolpidem (0.86 μg/mL), propofol (0.30 μg/mL), midazolam (0.08 μg/mL), thiopental (0.03 μg/mL), and amitriptyline (0.07 μg/mL). Adipose tissue and hair analysis suggested a previous and repeated use of these drugs verifying the fact that in Italy recreational abuse of anesthetic and sedative agents in health care practitioners is becoming an increasing problem.     

Postmortem Analysis of Electrogram Records from an Implantable Cardioverter‐Defibrillator (ICD) in the Reconstruction of a Road Traffic Accident

The case of a 69‐year‐old man, equipped with an ICD and suffering from several chronic cardiac diseases, who died in a car accident, was presented. We analyzed electrogram records from the ICD explanted from the body during the autopsy, which showed that the driver had suffered from malignant ventricular arrhythmia—ventricular fibrillation (VF). A thorough analysis of the details of the accident, as well as the timing of VF and the rhythm observed after the discharge of the ICD showed that the direct cause of the accident was the episode of arrhythmia resulting in a loss of consciousness. Therefore, the presented case illustrates the usefulness of postmortem analysis of electrogram records from ICDs in the reconstruction of road traffic accidents. In such cases, if the victims are implanted with ICDs, it should be a routine procedure performed by forensic pathologists.     

The Differentiation of Menstrual from Venous Blood and Other Body Fluids on Various Substrates Using ATR FT‐IR Spectroscopy

Crime scene investigators and laboratory analysts use chemical tests to detect and differentiate body fluids. Testing often requires a sample of the stain, and the chemicals may cause degradation of the fluid or interfere with subsequent tests. Colorimetric chemical tests do not differentiate between different types of the same fluid, such as venous and menstrual blood, and there is no presumptive test available to simultaneously differentiate several body fluids. In this study, we recorded ATR FT ‐IR spectra of venous and menstrual blood, semen, saliva, and breastmilk. Neat and simulated casework body fluid samples were analyzed on cotton, nylon, wood, paper, and glass substrates. Differences in fluid composition, including proteins and small molecules, resulted in spectral differences. Venous and menstrual blood is differentiated by the peak at 1039 cm^?1 attributed to phosphoric acid found in menstrual blood. Peak intensity is influenced by the porosity and weave of the substrate fabric.     

Penetration Depth of Projectiles Fired from a Replica of Colt Navy of 1851 in 20% Gelatin Blocks Correlated with Fatal Injuries Assessed in an Autopsy of a 78‐Year‐Old Man

Pursuant to the Polish Weapons and Ammunitions Law, muzzle‐loading black powder weapons, manufactured before 1885, or their replicas, as well as suitable projectiles, can be possessed legally without registration and special licenses. The aim of the study was to assess the penetration depth of projectiles fired from a black powder weapon, replica of Colt Navy of 1851, in 20% gelatin blocks and to compare the obtained results with the actual injuries found on autopsy of a 78‐year‐old man who had committed suicide. In the experiments, we used the black powder weapon and ammunitions, as well as gelatin blocks serving as a soft tissue model. We ascertained that solid spherical projectiles fired from black powder weapons cause extensive injuries, especially in the initial segment of the wound canal. Additionally, based on the presence and location of the wad in the wound canal, the distance from which the shot was fired can be determined.     

Postmortem Detection of Hepatitis B,C, and Human Immunodeficiency Virus Genomes in Blood Samples from Drug‐Related Deaths in Denmark*

Abstract: Blood‐borne viral infections are widespread among injecting drug users; however, it is difficult to include these patients in serological surveys. Therefore, we developed a national surveillance program based on postmortem testing of persons whose deaths were drug related. Blood collected at autopsy was tested for anti‐HBc, anti‐HBs, anti‐hepatits C virus (HCV), or anti‐human immunodeficiency virus (HIV) antibodies using commercial kits. Subsets of seropositive samples were screened for viral genomes using sensitive in‐house and commercial polymerase chain reaction (PCR) assays. Hepatitis B virus (HBV) DNA was detected in 20% (3/15) of anti‐HBc‐positive/anti‐HBs‐negative samples, HCV RNA was found in 64% (16/25) of anti‐HCV‐positive samples, and HIV RNA was detected in 40% (6/15) of anti‐HIV‐positive samples. The postmortem and antemortem prevalences of HBV DNA and HCV RNA were similar. Postmortem HIV RNA testing was less sensitive than antemortem testing. Thus, postmortem PCR analysis for HBV and HBC infection is feasible and relevant for demonstrating ongoing infections at death or for transmission analysis during outbreaks.     

Stability of Morphine,Codeine, and 6‐Acetylmorphine in Blood at Different Sampling and Storage Conditions

The stability of drugs in biological specimens is a major concern during the evaluation of the toxicological results. The stability of morphine, codeine, and 6‐acetyl‐morphine in blood was studied after different sampling conditions: (i) in glass, polypropylene or polystyrene tubes, (ii) with addition of dipotassium ethylene diamine tetraacetic acid (K₂EDTA) or sodium oxalate (Na₂C₂O₄), and (iii) with or without the addition of sodium fluoride (NaF). Spiked blood samples were stored at two different temperatures (4 and ?20^°C), analyzed after different storage times and after three freeze–thaw cycles. Opiate concentrations were decreased in all conditions, but the most unstable was 6‐acetyl‐morphine. The addition of NaF as preservative improved the stability of opiates at all conditions studied, whereas the type of anticoagulant did not affect the stability of opiates. It was concluded that blood samples should be stored at ?20^°C in glass tubes containing oxalate and NaF for maximum stability.