Biochemical blood markers and sampling sites in forensic autopsy

Forensic pathologists often hesitate to use biochemical blood markers due to the risk of large postmortem changes and deviations from healthy subjects. Biochemical analyses of postmortem blood, if possible, may help to evaluate pathological status and determining the cause of death in forensic diagnosis, for example, in sudden unexpected death without obvious cause, or young adults with no apparent cause of death or antemortem information. Even commercially available biochemical markers were re-evaluated in the blood samples of 164 forensic autopsy cases. Biochemical markers examined were HbA1c, fructosamine, blood nitrogen urea (BUN), creatinine, total protein, total bilirubin, gamma-glutamyl transpeptidase (gamma-GTP), triglyceride, total cholesterol, C-reactive protein (CRP) and pseudocholine esterase (pChE). We collected cardiac blood (left cardiac blood and right cardiac blood) and peripheral blood (femoral vein blood) to clarify the differences in measured values by sampling site. The measured values were analyzed in relation to postmortem interval, etiology of death and sampling sites. Of all eleven markers, HbA1c is the most useful and reliable because of its negligible postmortem changes and small deviation from healthy subjects. Total bilirubin, BUN, CRP and total cholesterol can be useful if we set appropriate limit ranges and pay attention to the interpretation. For the evaluation of changes due to postmortem intervals, none of the markers except for triglyceride showed significant changes up to three days postmortem. As for sampling sites, femoral vein blood is generally recommended considering postmortem changes, but left cardiac blood was suitable for creatinine, pChE, and total cholesterol. For clinical forensic diagnosis of biochemical blood markers, we must determine the "forensic abnormal value" after collecting more cases by known causes with more information about the population.     

Investigation of oxyhemoglobin and carboxyhemoglobin ratios in right and left cardiac blood for diagnosis of fatal hypothermia and death by fire

Few large-scale investigations have looked at the oxyhemoglobin ratio (%O₂-Hb) or the carboxyhemoglobin ratio (%CO-Hb) in fatal hypothermia and death by fire as applicable to forensic medicine. We therefore retrospectively examined right and left cardiac blood samples for both %O₂-Hb and %CO-Hb in 690 forensic autopsy cases. We therefore sought to establish reference values for the above forensic diagnoses, to compare %O₂-Hb in fatal hypothermia with or without cardiopulmonary resuscitation (CPR), and to compare the relationship between %CO-Hb and smoking history. All %O₂-Hb and %CO-Hb data were obtained during or immediately after autopsies using a portable CO-oximeter. Death by carbon monoxide (CO) intoxication and death by fire were excluded from the analysis involving smoking history.In fatal hypothermia, %O₂-Hb in the left cardiac blood was significantly higher than that in the right cardiac blood, providing important evidence for fatal hypothermia. Furthermore, %O₂-Hb in the left cardiac blood increases with CPR but that in the right cardiac blood increases in parallel. No correlation was observed between rectal temperature and %O₂-Hb in the right and left cardiac blood, indicating that it is unlikely that postmortem cooling increases %O₂-Hb in cardiac blood. %CO-Hb in smokers was significantly higher than that in non-smokers, although the number of cigarettes smoked did not appear to be significant. When assessing death by fire, we identified that %CO-Hb of >10% was a reliable marker of antemortem CO inhalation, regardless of smoking history.     

Ethyl sulfate in blood shows the potential to distinguish alcoholic death and postmortem alcohol instillation

Alcohol is often found in the blood of the deceased. To cover up the true cause of victim’s death, postmortem instillation of alcohol occurs in some criminal cases. Explaining the finding of alcohol is extremely vital in forensic practice. This study aims to evaluate whether ethyl glucuronide (EtG) and ethyl sulfate (EtS) in blood and vitreous humor (VH) can be used to distinguish alcoholic death and postmortem alcohol instillation. Saline or 12.6 g/kg ethanol (antemortem alcohol poisoning group) was introduced into rabbits’ stomachs 2 h before sacrificed. Same amount of ethanol was introduced into rabbits’ stomachs at 0 h, 0.5 h, 1 h and 2 h after death in four subgroups of postmortem alcohol instillation group, respectively. Cardiac blood and VH were collected at 10 min, 4 h, 10 h and 24 h after death in blank and antemortem alcohol poisoning group, and after instillation of alcohol in postmortem alcohol instillation group. Blood was also collected at 34 h. Ethanol and EtG levels in blood and VH and EtS in VH in antemortem alcohol poisoning group were overlapped with those in postmortem alcohol instillation group. The contents of EtG and EtS in blood in antemortem alcohol poisoning group (mean ≥ 7.833 μg/mL for EtG and ≥ 19.990 μg/mL for EtS) were much higher than those in postmortem alcohol instillation group (mean ≤ 0.118 μg/mL for EtG and ≤ 0.091 μg/mL for EtS), but apparent decomposition was observed in EtG, which might lead to misinterpretation. Blood EtS showed better stability and could be used to distinguish alcoholic death and postmortem alcohol instillation.     

Is acute subdural hematoma reduced during the agonal stage and postmortem?

The issue of proper use of postmortem computed tomography (PMCT) in forensic fields is currently being actively discussed. The PMCT image has specific findings that differ from the antemortem image, and it is essential to understand and interpret postmortem changes in order to utilize PMCT properly. In this article, we present two cases of acute subdural hematoma (ASDH) in which images were obtained both ante- and postmortem. These images showed marked reduction of hematoma and diminishing midline shift between the agonal and postmortem periods, without evacuation of the hematoma. Attention should be paid to this phenomenon because key findings in determining cause of death could disappear if investigating the cause of death takes too long in cases that prove to be ASDH. In other words, this phenomenon potentially becomes a risk for misdiagnosis when we decide the cause of death without knowing the details of the circumstances of death.     

Microbial community analyses provide a differential diagnosis for the antemortem and postmortem injury of decayed cadaver: An animal model

Differentiating antemortem injury from postmortem injury of decayed cadavers is one of the difficult issues in forensic science. Forensic pathologists identify antemortem injury according to the macroscopic and microscopic vital reactions taken place after being injured. However, the decomposition would render those vital reactions ineffective. Microbiomes have been widely used in forensic science due to their succession with time and sensitivity to vary of environment. In this study, microbiomes were introduced to determine whether the bacterial communities can be used to distinguish between the ante- and postmortem injuries through an animal experiment. Our findings showed that the differences of bacterial community were increasingly apparent from the 6th to 9th day after the wound created when the types of wounds were unidentified by morphological examination due to decomposition. The biomarkers at the genus level could effectively distinguish between injury types, Among them, Enterococcus and Enterobacter were only observed in the antemortem injured group, while Staphylococcus and Acinetobacter were only in the postmortem injured group. It is possible to tell whether cadaveric injuries developed before or after death by detecting differences in the bacterial communities of putrefying wounds. This study provides a new perspective for the differences between ante- and postmortem injuries and provides a promising method for us to identify the ante- and postmortem wounds, especially in decomposed cadavers.     

Eifeler Armbrust-Fall

In most instances external postmortem examinations of corpses are done unprofessionally and without forensic experience. Important indications for homicide are often overlooked. Nevertheless, even if they are done accurately external postmortem examinations cannot replace forensic autopsies. Only an autopsy will prove definite results concerning the cause of death and only they can exclude homicide. In the following article the murder of a young man with a crossbow is described which almost would not have been discovered due to the mentioned weak points.     

Is the formula of Traub still up to date in antemortem blood glucose level estimation?

According to the hypothesis of Traub, also known as the ‘formula of Traub’, postmortem values of glucose and lactate found in the cerebrospinal fluid or vitreous humor are considered indicators of antemortem blood glucose levels. However, because the lactate concentration increases in the vitreous and cerebrospinal fluid after death, some authors postulated that using the sum value to estimate antemortem blood glucose levels could lead to an overestimation of the cases of glucose metabolic disorders with fatal outcomes, such as diabetic ketoacidosis. The aim of our study, performed on 470 consecutive forensic cases, was to ascertain the advantages of the sum value to estimate antemortem blood glucose concentrations and, consequently, to rule out fatal diabetic ketoacidosis as the cause of death. Other biochemical parameters, such as blood 3-beta-hydroxybutyrate, acetoacetate, acetone, glycated haemoglobin and urine glucose levels, were also determined. In addition, postmortem native CT scan, autopsy, histology, neuropathology and toxicology were performed to confirm diabetic ketoacidosis as the cause of death. According to our results, the sum value does not add any further information for the estimation of antemortem blood glucose concentration. The vitreous glucose concentration appears to be the most reliable marker to estimate antemortem hyperglycaemia and, along with the determination of other biochemical markers (such as blood acetone and 3-beta-hydroxybutyrate, urine glucose and glycated haemoglobin), to confirm diabetic ketoacidosis as the cause of death.     

Urinary phenylacetylglutamine as a possible biomarker for central nervous system disorders in forensic autopsy cases

Phenylacetylglutamine (PAG) is a metabolite that is excreted in human urine. Phenylalanine is metabolized to phenylacetic acid, which is then amide-bonded to glutamine to form PAG. We are currently studying PAG as a urinary biomarker in forensic autopsy cases.Materials and MethodsUrine samples were collected from 188 forensic autopsy cases and the urinary PAG concentration was analyzed quantitatively using GC-MS. Urinary creatinine (Cr) concentration was also analyzed by GC-MS. For statistical analysis, the JMP Pro 15.0.0 software program was used. The relationship between urine PAG/Cr (the ratio of each concentration), sex, age, postmortem interval (PMI), survival duration, and cause of death was statistically analyzed.Results and DiscussionThe median (range) of PAG/Cr was 0.12 (0.002-3.26). The PAG/Cr ratio showed no significant relationship to sex or survival duration. Regarding the cause of death, traumatic brain injuries had a significantly higher ratio than intoxication (p=0.023). Cerebrovascular disease, such as cerebral hemorrhage and subarachnoid hemorrhage, did not differ significantly from any cause of death group. However, when traumatic brain injuries and cerebrovascular accidents are combined as one cause of death group, the PAG/Cr value of CNS damages was significantly higher than that of intoxication (p=0.062).ConclusionUrinary PAG/Cr might be a biomarker not only for traumatic brain injuries but also for antemortem CNS damages.     

Starch accumulation in the lungs of two infants following positive ventilation

Two infants in different nurseries were found with cardiopulmonary arrest. Cardiopulmonary resuscitation was undertaken immediately in both cases, but was unsuccessful. The cause of death in both infants was diagnosed as sudden unexpected death, probably sudden infant death syndrome, at postmortem investigations. Microscopic examination at autopsy showed the presence of starch granules in the lungs. These were probably introduced during tracheal or nasotracheal intubation for cardiopulmonary resuscitation from gloves sterilized with powdered cornstarch. In both cases cellular staining of foreign bodies was weak and there were few starch granules within macrophages. Our findings suggest that the detection of cornstarch in the lungs can be an artifact arising from surgical gloves used in resuscitation. This artifact may easily occur in infants because of their immature lungs and short respiratory tract. Non-powdered gloves should be worn instead of powdered gloves during tracheal intubation, especially in infants.     

Investigation of tuberculosis prevalence by acid-fast stain,culture and real-time PCR method in forensic autopsies

Tuberculosis (TB) is still one of the most important diseases and causes of death in the world. We aimed to investigate TB prevalence in forensic autopsy cases in Turkey by the acid-fast bacilli (AFB) staining, TB culture and real-time polymerase chain reaction (RT-PCR) methods in paraffin-embedded tissues. From January 2012 to January 2015, 1676 tissue samples were examined under AFB staining and TB culture, and 85 paraffin-embedded tissue samples under RT-PCR from 14,083 prospectively-investigated forensic autopsy cases in the Istanbul Council of Forensic Medicine. Positivity with microbiological methods (AFB staining and/or TB culture and/or RT-PCR) was detected in 43 (0.3%) out of 73 (0.5%) cases in which TB was diagnosed with histopathological findings. Acid-fast bacilli were detected in 14 tissue samples by AFB staining, and a total of 11 tissue samples were found culture-positive in the Löwenstein-Jensen medium. Mycobacterium tuberculosis DNA was positive in 36 samples with RT-PCR. Eleven (15%) cases were detected as miliary tuberculosis. Microbiological sampling should accompany autopsy findings and histopathological sampling for post-mortem identification and notification of active TB during forensic autopsies. Diagnosis of TB in forensic autopsies would contribute to clarifying the cause of death and to the collection of epidemiological data in Turkey, particularly in ante-mortem undiagnosed cases.     

Effects of postmortem positional changes on conjunctival petechiae

The present study aimed to determine whether postmortem period, physical constitution, cause of death, and cardiopulmonary resuscitation are associated with positional changes in the postmortem appearance of conjunctival petechiae. We retrospectively investigated serial forensic autopsies from 6 h to 6 days postmortem (n = 442; male, 303; female, 139; median age, 62 years; range, 0–100 years). The causes of death were sharp instrument injury, blunt force trauma, fire, intoxication, asphyxia, drowning, hypothermia, hyperthermia, acute heart failure, and natural causes. Of these, 28 (male, n = 18; female, n = 10) were aged ≤5 years. Palpebral conjunctival petechiae were initially assessed at autopsy in supine bodies, then reassessed in prone bodies after 30 min. Among 414 bodies, 291 (70.2%) and 123 (29.7%) who were aged between 6 and 100 years, and 18 (64.2%) and 10 (35.7%) aged <5 years at the time of death, were discovered in the supine and prone positions, respectively. The amounts of petechiae increased within 1.5 days postmortem, but not in those discovered in the prone position. The rates at which petechiae increased were higher in supine overweight bodies (BMI ≥ 25.0) and in those who were discovered supine and had died of asphyxia or drowning (37.5%). Cardiopulmonary resuscitation for bodies discovered in the supine and prone positions did not statistically affect the occurrence of petechiae. Several postmortem factors can cause hypostatic blood redistribution that manifests as increased amounts of petechiae in the palpebral conjunctivae.     

Postmortem changes in serum noradrenaline and adrenaline concentrations in rabbit and human cadavers

Postmortem changes in serum noradrenaline and adrenaline concentrations in adrenectomised rabbits and in human sudden death cases were measured with HPLC in order to obtain information of the usefulness of these biogenic amines as indicators of antemortem stress. It appeared that serum concentrations increased with time postmortem, except for adrenaline in the adrenectomised rabbits. The values varied considerably between individuals in both materials. It is concluded that postmortem assays of serum catecholamines cannot be used in practice to demonstrate antemortem stress, although this would theoretically be a promising approach, e.g. in cases of suffocation. So far there seems to be no reliable and conclusive method for estimating short lasting antemortem stress in individual forensic cases.     

Forensic diagnosis of ante- and postmortem burn based on aquaporin-3 gene expression in the skin

In order to diagnose death associated with fire, it is essential to show that the person was exposed to heat while still alive. We investigated both AQP1 and AQP3 expression in the skin of an experimental burn model, as well as in forensic autopsy cases, and discuss its role in the differential diagnosis of ante- and postmortem burns. In animal experiments, there was no difference in AQP1 gene expression among four groups (n = 4): antemortem burn, postmortem burn, mechanical wound, and control. However, AQP3 expression in the antemortem burn was increased significantly compared with that of the other groups even at 5 min after burn. Water content of the skin was decreased significantly by the burn procedure. Consistent with animal experiments, AQP3 gene expression in the skin of antemortem burn cases was increased significantly compared with postmortem burns, mechanical wounds, and controls (n = 12 in each group). These observations suggest that dermal AQP3 gene expression was increased to maintain water homeostasis in response to dehydration from burn. Finally, our results suggest that AQP3 gene expression may be useful for forensic molecular diagnosis of antemortem burn.     

Immunohistochemical evaluation of hypoxia markers in the myocardium

Some proteins, termed hypoxia markers, are more highly expressed in hypoxic cells including ischemic cells. We evaluated the timing of the expression of various hypoxia markers and their specificity of expression in the myocardium in various causes of death. We selected two cases for each cause of death from the forensic autopsies performed by our department and evaluated the diffuse expression of hypoxia markers in the myocardium. We also evaluated the focal expression of these markers in the ventricles of five sudden cardiac death cases. We found that the expression of hypoxia markers in the myocardium varied depending on the cause of death and length of the dying process. This information may help in determining the cause of death in forensic medicine. In particular, we identified RBM3 and CCC9 as markers for ischemic heart disease. Immunohistological staining of myocardium with hypoxia markers could help to find the cause leading to death.     

Forensic aspects of complications resulting from cardiopulmonary resuscitation

While cardiopulmonary resuscitation (CPR) can save lives, it can also injure patients. As a result, forensic pathologists often see CPR-related injuries during autopsies that are unrelated to the patients' primary cause of death. Therefore, pathologists must be able to distinguish between CPR-related injuries and those caused by other factors, such as assaults or accidental violence. This distinction is complicated because even therapeutically unimportant injuries can have forensic significance. For example, resuscitative injuries are observed frequently in the neck and the chest. This article focuses mainly on complications due to ventilation and chest compression during CPR. The following iatrogenic complications are described: bruising and abrasions in the face and neck, fractures of the hyoid bone and thyroid cartilage, air way injuries, vomitus aspiration, positional error of the tube for intra-tracheal intubation, petechiae, retinal hemorrhages, subarachnoid hemorrhages, rib and sternum fractures, bone marrow embolism, cardiac injuries including myocardial hemorrhages and frothy heart blood, and injuries to the abdominal organs such as liver and spleen.     

Weight ratio of the lungs and pleural effusion to the spleen in the diagnosis of drowning

An accurate and reliable method of diagnosing death by drowning is an important requirement in forensic autopsies. In this study, we compared the weight ratio of the lungs and pleural effusion to the spleen for 55 cases of drowning (37 males, 18 females), 36 cases of mechanical asphyxiation (16 males, 20 females), and 26 cases of acute cardiac death (19 males, 7 females). In the case of the males, there were significant differences in the weight of the spleen and the total weight of the lungs and pleural effusion between drowning and the other causes of death; however, there was no such significant difference in the females. We observed significant differences in the lungs and pleural effusion/spleen weight ratio between drowning and the other causes of death for both sexes. Therefore, these findings suggest that the ratio may be a useful index to accurately diagnose death by drowning, while ruling out mechanical asphyxiation and acute cardiac death in forensic autopsies.     

Immunohistochemical expression of tumor necrosis factor-α in sepsis-induced lung injury

Sepsis is asevere, systemic inflammatory disease caused by various kinds of microbes. In the present study, we immunohistochemically examined tumor necrosis factor (TNF)-α expression in sepsis-induced lung injury, and discuss its availability for the postmortem diagnosis of sepsis. Lung samples were obtained from different lung lobes of nine sepsis and eight control cases with postmortem intervals between 12 and 48 hours. Immunohistochemical analysis using anti-human TNF-α rabbit polyclonal antibodies was carried out. In sepsis and control groups, immunoreactivity for TNF-α was strongly detected in round-shaped mononuclear cells. The intensity of the immunohistochemical staining reaction was homogeneous in all lobes of the lungs examined. Furthermore, a double-color immunofluorescence analysis revealed that macrophages were a main cellular source of TNF-α in the lungs. To semiquantitatively evaluate the expression of TNF-α in the lungs, the ratios of the number of TNF-α-positive macrophages to total number of macrophages were calculated. Morphometrically, in lungs of the sepsis group, the ratio of TNF-α-positive macrophages was significantly higher, compared with the control group. TNF-α expression in the lungs can become a clue for the postmortem diagnosis of pulmonary inflammation, especially, TNF-α-positive ratios of 20% of more might suggest sepsis as the cause of death. This study was presented at the Sixth International Symposium in Advanced Legal Medicine (ISLAM), Hamburg, Germany, September 2006     

Temporal evolution in peritoneal hemorrhage as depicted by postmortem CT

BackgroundComputed Tomography (CT) is now utilized as an effective tool for postmortem diagnoses. However, reports on the temporal evolution in imaging findings of these postmortem CT is limited.Case presentationOne night, a 66 year old male following drinking at a bar on his way home slept in the road. He was ran over by a taxi at 01:28 and immediately transferred to a nearby hospital. During his transfer to the hospital, he lost consciousness and fell into a state of cardiopulmonary arrest, and died despite resuscitation attempts. CT images were taken at 02:30. Following this, CT images were re-taken 54 h after death, just prior to a medicolegal autopsy.Result & discussionSmall amount of intra-abdominal hemorrhage was found during the initial CT examination. However, the extent of intra-abdominal bleeding observed during the second CT examination performed 54 h later had substantially increased. During the autopsy, the amount of intra-abdominal hemorrhage was 1700 mL. Injury to the mesentery, liver and pancreas was also observed. Additional major injuries discovered during the autopsy were, skin abrasions and lacerations of the scalp, subarachnoid hemorrhage, fractures of the ribs, right humerus, and pelvic bones. The deceased postmortem blood alcohol level was 2.4 mg/mL. The cause of death was determined as exanguination due to systemic injury. The mechanism of the postmortem increase in the intra-abdominal hemorrhage remained unknown. However, the amount of bleeding found during autopsies may not be the same as that at the time of death. As a result, the criteria for the diagnosis of the cause of death in autopsies should be carefully reconsidered. In addition, accompanying CT imaging at the time of death and possible postmortem changes should be carefully considered in postmortem CT imaging, so that there will not be incorrect assignment of the causes of death.     

Resuscitation and conjunctival petechial hemorrhages

In recent years, cardiopulmonary resuscitation (CPR) has been discussed as a cause of petechial hemorrhage in eyelids and conjunctivae, which could be of substantial significance to forensic expertises in cases of suspected strangulation. In the reported series or case observations, the combination of CPR and petechiae seemed to be sufficient to explain such a causal connection. Nearly all presented cases were victims for which the mechanisms resulting in death were themselves well-known causes explaining the development of such petechiae; and said mechanisms can frequently be observed in victims that did not receive CPR. An earlier, also retrospective, analysis of a series of forensic autopsies did not confirm CPR as a significant cause of conjunctival petechiae. Now we present the result of a prospective examination of 196 resuscitations of adult patients with separate assessment of petechiae being present even prior to resuscitation. Petechiae were present in 12 cases – but in eight of them prior to resuscitation already. Three other persons with petechiae found only after CPR were in the group of successfully resuscitated persons and exhibited petechiae hours after CPR during therapy in intensive care units – during a phase of ongoing cardiac insufficiency, which obviously caused them. The only case with petechiae observed neither immediately prior to nor after unsuccessful resuscitation, but during a follow-up examination one day later, needs to be discussed. It is not interpreted as reliable evidence for the causality of CPR though. Our interpretation of reports in literature as well as our experiences confirm the absence of actual proof of petechiae being generated by CPR and in the presence of generally significant doubts of this relation.     

FVIIIra, CD15, and tryptase performance in the diagnosis of skin stab wound vitality in forensic pathology

The timing of skin wounds is one of the most challenging problems in forensic pathology. In the first minutes or hours after infliction, histological examination fails to determine whether a wound was sustained before or after death. The aim of this study was to evaluate the use of three immunohistochemical markers (FVIIIra, CD15, and tryptase) for the interpretation of the timing of cutaneous stab wounds. We evaluated these markers in intravital wounds from autopsy cases (n?=?12) and surgical specimens (n?=?58). As controls, we used normal skin samples from autopsies (n?=?8) and an original ex vivo surgical human model of recent postmortem wounds (n?=?24). We found overexpression of FVIIIra in 100 % of vital wounds, but also in 53 % of the controls. The number of CD15-positive cells was higher in wound margins than in internal controls (p?<?0.0001) and was significantly correlated with the time interval between incision and devascularization (p?=?0.0005; minimal time for positivity, 9 min). Using the anti-tryptase antibody, we found that the mast cell degranulation rate was higher in wound margins (p?<?0.0001) and correlated with the time interval (minimal time, 1 min). The sensitivity and specificity for the diagnosis of vitality were respectively 100 and 47 % for FVIIIra, 47 and 100 % for CD15, and 60 and 100 % for tryptase. The inter-observer agreement coefficients were 0.68 for FVIIIra, 0.90 for CD15, and 0.46 for tryptase. Finally, we demonstrated that these markers were not reliable in putrefied or desiccated specimens. In conclusion, CD15 and tryptase, but not FVIIIra, may be useful markers for differentiating recent antemortem from postmortem injuries.