The forensic-biochemical diagnosis of different-genesis hypoxic conditions in cadavers of newborns and babies

Some parameters of anaerobic glycolysis were examined in trauma and hypoxia of different types. Statistically reliable differences were detected in the level of glucose in blood, of glycogen and of lactic acid as well as in the activity of lactatodrogenases in the liver and cardiac and skeleton muscles as observed in different-type hypoxia versus trauma. Diagnosis criteria were designed for the diagnosis of hypoxic conditions in newborns and babies.     

Sudden infant death and liver phosphoenolpyruvate carboxykinase analysis

In an effort to substantiate the impaired gluconeogenesis-terminal hypoglycemia hypothesis of sudden infant death syndrome (SIDS), 52 infants ranging from 3 weeks to 7 months of age which had been brought to autopsy were studied. The stomach contents, vitreous humor glucose concentrations, hepatic glycogen content and hepatic phospho-enolpyruvate carboxykinase (PEPCK) activity were measured as part of the laboratory component of the postmortem investigation. The stomach contents, vitreous humor glucose concentrations and liver glycogen content were similar in SIDS/and non-SIDS victims. PEPCK activity was, however, significantly lower in SIDS (p < 0.001) victims and in SIDS with other findings (p < 0.01) victims when compared to non-SIDS victims. Despite the fact that SIDS victims had lower hepatic PEPCK activity and hence potentially lower gluconeogenic capacity, terminal hypoglycemia could not be demonstrated in this group as compared to the SIDS with other findings and the non-SIDS infants. The impaired gluconeogenesis-terminal hypoglycemia hypothesis thus could not be substantiated.     

Hepatic glycogen staining. Applications in injury survival time and child abuse

Hepatic glycogen stores have long been known to decrease with starvation, trauma, acute stress, and shock. In this study, hepatic tissue was examined in 122 decedents ranging in age from 1 to 88 years who died of a variety of causes. Hepatic tissue was stained for glycogen/carbohydrates using the Best's carmine and/or period acid-Schiff (PAS)-alcian blue methods in 121 cases. The liver samples were evaluated for amount of staining, staining around hepatic injuries (if present), and presence of histologic abnormalities. Prominence of staining was decreased with increasing survival times after injury (P=.0001) and increasing postmortem interval. Staining along the edges of hepatic injuries was decreased in antemortem injuries but not in postmortem-induced injuries (P<.0001). If used in conjunction with circumstantial and autopsy information, hepatic glycogen/carbohydrate staining using the Best's carmine and PAS-alcian blue methods could greatly aid in the determination of injury survival times and the vital nature of questionable injuries in child deaths and other cases.     

Efficacy of cerebro-spinal fluid biochemistry in the diagnosis of brain insult.

Postmortem biochemical indices may provide a useful adjunct to morphological studies in the identification of antemortem brain insult. We studied 34 routine medico-legal cases categorising them into one of four diagnostic groups. There were 11 cases of head trauma, 7 of 'hypoxia' (3 hangings and 4 carbon monoxide or drug poisonings), 7 sudden cardiac deaths and 9 miscellaneous cases. Survival time and postmortem interval was known for each case. The degree of cranio-cerebral trauma was graded. Cerebro-spinal fluid (CSF) and vitreous humour were analysed for calcium, glucose, total proteins, aldolase, aspartate transaminase (AST), alanine transaminase (ALT), gamma glutamyltransferase (GGT), lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase BB isoenzyme (CK-BB). CK-BB was also measured in superior vena cava serum. In CSF there was a significant correlation between the severity of cranio-cerebral trauma and levels of aldolase, CK-BB, AST, ALT and total proteins. CSF CK-BB, median units/l (range), for the groupings of head trauma, hypoxia, sudden cardiac death and miscellaneous were respectively 823 (2-3431); 96 (2-187); 4 (2-25); 5 (1-69). Corresponding serum CK-BB levels were 240 (28-322); 390 (26-411); 180 (20-482); 79 (18-530).     

Lugol's test reexamined again: buccal cells

Lugol's iodine staining technique was used to examine oral samples from 10 men and 10 women. Examination of saliva samples before and after extraction with water shows that the low levels (49 positive cells and 3,951 negative cells) of glycogen in buccal epithelial cells become even lower after water extraction (0 positive cells and 4,000 negative cells). In addition to the 20 samples used in this paper, 40 oral swabs extracted with water were examined under classroom conditions with much less than 1% of the epithelial cells being positive for glycogen. Furthermore, 119 saliva samples from chewed gauze in sexual assault kits were extracted with water and all of them yielded less than 1% glycogen positive cells. This paper proposes that when more than 1% of the nucleated squamous epithelial cells are glycogen positive with Lugol's test after extraction in water, it is reasonable to eliminate the mouth as a source of these glycogen positive cells.     

Optimization of DNA extraction from low-yield and degraded samples using the BioRobot EZ1 and BioRobot M48

Robotic extraction of DNA from dilutions of blood and semen using either the BioRobots EZ1 or BioRobots M48 consistently produced lower recoveries than standard organic extractions of the same samples. In an effort to increase the efficiency of robotically extracted DNA, glycogen and carrier RNA were added following cell lysis. The addition of glycogen, postlysis, resulted in no improvement in DNA recovery with the BioRobot EZ1. However, when carrier RNA was added to the cell lysate of limited and degraded samples extracted on the EZ1 or the M48, DNA recoveries dramatically increased four- to 20-fold. DNA yields obtained by robotic extraction in the presence of carrier RNA were as high, or higher, as those obtained by organic extraction lacking carrier RNA, while experiments that utilized carrier RNA in both types of extractions showed increased sensitivity for both methods. Furthermore, carrier RNA substantially increased the recovery of fragmented DNA with the EZ1.     

Hyperosmolarity Induces Armanni‐Ebstein‐like Renal Tubular Epithelial Swelling and Cytoplasmic Vacuolization

Armanni–Ebstein lesions have been considered pathognomonic for diabetes mellitus and appear as markedly swollen renal tubular epithelial cells with cytoplasmic clearing and glycogen accumulation. However, the extent to which hyperosmolarity contributes to the Armanni–Ebstein phenotype is unclear. Ten sheep were injected intravenously with 20% mannitol at 11 mOsm/kg, and subsequent histological evaluation of the kidneys showed variable degrees of osmotic nephrosis and cytoplasmic clearing of renal tubular epithelial cells similar to that seen with Armanni–Ebstein lesions. However, although morphological changes similar to Armanni–Ebstein lesions could be produced, no intracytoplasmic glycogen was demonstrated with periodic Acid–Schiff (PAS) stain. This suggests that while hyperosmolarity may contribute to the development of an Armanni–Ebstein phenotype, glycogen accumulation may result from the more complex metabolic effects of glucose on renal tubular epithelial cells. Thus, when Armanni/Ebstein‐like vacuolizations are seen at autopsy, a confirmatory PAS stain is recommended because of the potential effect of hyperosmolar states.     

Serum beta-glucuronidase activity in polytrauma patients and in centrifuged autopsy blood samples]

The serum activity of beta-glucuronidase was investigated in 58 patients after severe trauma as well as in 43 autopsy cases. In 10 cases the enzyme activities in postmortem blood samples from the femoral vein were compared to those present in the correspondent heart blood samples. An elevated activity of beta-glucuronidase was observed in 14% of the patients within the first 36 h after severe trauma increasing to 62% in blood samples collected later on. The activity of beta-glucuronidase in the heart blood samples was always higher than in the corresponding sample from the femoral vein. In cases of prolonged post-mortem interval an elevated activity might have been due to bacterial contamination. In postmortal blood samples from the femoral vein an elevated enzyme activity was found in 70% of the study material. The results of the preliminary study on the activity of beta-glucuronidase in blood samples frequent in forensic routine work indicated that an elevated enzyme activity might be present for the following scenery: after severe trauma, in alcohol/drug abuse, presence of putridity/autolysis, presence of inflammatory processes, in diabetes as well as in carcinoma diseases. The significance of elevated beta-glucuronidase activity concerning alterations of unconjugated drug concentration due to in vitro cleavage of O-glucuronides should be investigated.     

Computer analysis of erythrocyte catalase activity in diagnosis of mild brain trauma and concussion

A complex investigation of catalase activity in erythrocytes was conducted basing on the results of computer quantitative morphometry of histochemical examination of blood samples from persons with mild craniocerebral trauma. It was found that erythrocytic catalase activity correlates with severity of brain trauma. A novel technique of objective histochemical diagnosis of trauma is described. It provides objective grounds for expert conclusions. Differential features of erythrocytic shape were defined in a small series of micropreparations vs the rest blood samples.     

Postmortem metabolic indices of antemortem adrenergic stimulation

Tissue lactate concentration has been reported to be a useful postmortem indicator of antemortem awareness of mortal danger. The purpose of this study was to determine further whether selected tissue metabolites could be used as postmortem markers of antemortem adrenergic stress. Sprague-Dawley albino rats were anesthetized with pentobarbital and then injected with 2.0 mg kg-1 i.p. epinephrine hydrochloride to induce experimentally a severe sympathetic response that may be associated with the awareness of mortal danger; 20 min after the injection of epinephrine, when the metabolic response was at its peak, the animals were killed by exsanguination. Samples of the following tissues were removed immediately prior to death (0 h) and 48 h postmortem: soleus, plantaris, kidney medulla, kidney cortex, liver, and heart. These samples were analyzed for glycogen, lactate, ATP, creatine phosphate, pH, and total protein concentration. Significant differences in lactate concentration were observed in all tissues except soleus at 0 h in the epinephrine-injected animals. Specific tissues also had significant reductions in glycogen, ATP, and creatine phosphate concentrations at 0 h. At 48 h postmortem, however, only the liver and soleus lactate concentrations were significantly different from the 48-h control samples. It is unlikely that these small differences found in some tissues at 48 h postmortem would be detected in an uncontrolled accident situation. We concluded from these findings that these selected tissue metabolites are not useful as long-term postmortem indicators of antemortem adrenergically induced hypermetabolism.     

二维红外光谱用于急性心肌缺血猝死的鉴别研究

目的采用衰减全反射-傅里叶变换红外光谱(ATR-FTIR)技术检测急性心肌缺血猝死大鼠的血清分子特征,探讨二维红外光谱鉴别急性心肌缺血猝死的法医学价值。方法成年雄性SD大鼠随机分为急性心肌缺血猝死组(实验组)和空气栓塞死组(对照组),血清样本行ATR-FTIR检测,应用OMNIC软件进行光谱预处理并求二阶导数光谱,分别计算两组原始光谱及二阶导数光谱的相似度,并应用Matlab R2010a软件建立二维光谱模型。结果实验组与对照组的大鼠血清的原始红外光谱在峰形、峰位、峰强度均极其相似,相似度达99.78%,二阶导数光谱显示出两者相似度降至98.62%,二维光谱模型显示两组光谱在1 625cm~(-1)、1 550cm~(-1)、1 080cm~(-1)及860cm~(-1)附近的成分差异,且它们的自动峰个数不相同,提示两组在蛋白质、糖原、核酸等分子含量存在较明显差异。结论仅从一维光谱上难以区分出两组的分子差异,二维红外光谱可以直观、有效地反映出急性心肌缺血猝死与空气栓塞死的大鼠血清分子差异,有望应用于法医学死因鉴别。     

Aminopeptidase and cathepsin A activity in vitreous humor in relation to causes of death

Brain autolysis happens rapidly, especially when environmental temperatures are high, and poses serious limitations for evaluating damage using morphologic methods. In the present study we have measured total proteins, cathepsin A and aminopeptidase activity in the vitreous humor in relation to cause of death and survival time. We have studied vitreous humor samples from 106 cadavers autopsied in the I.A.F. of Granada. The samples were classified according to causes of death as follows: myocardial infarction; hanging; other mechanical asphyxias; multiple trauma; craniocerebral trauma; other violent death; pulmonary functions; and other natural deaths. Total protein was measured by Lowry's method, and cathepsin A and aminopeptidase activity by the Bowen and Davison (1973) and Greenberg (1962) methods, respectively. Results are expressed in IU/l and in mIU/mg of protein. We found higher values of aminopeptidase and cathepsin A activity in groups with severe brain damage (craniocerebral trauma, multiple trauma, etc.) and lower values in groups of natural deaths. We believe, therefore, that aminopeptidase activity in vitreous humor may be a useful parameter for evaluating brain damage.     

大鼠死后腓肠肌内糖原含量变化与死亡时间的关系

目的研究大鼠死亡后腓肠肌内糖原含量变化与死亡时间之间的关系。方法采用断颈、机械性窒息、失血性休克三种方法分别处死大鼠,于死亡后0﹑0.5﹑1﹑1.5﹑2﹑3﹑4﹑8﹑12﹑18和24h时间点取腓肠肌,利用PAS染色方法并结合计算机图像分析技术分析糖原含量变化。结果大鼠腓肠肌内糖原含量和阳性面积随死亡后时间的延长而呈线性减少。结论腓肠肌内糖原含量和阳性面积可作为死亡时间推断的参考指标。     

The diagnostic value of postmortem blood glucose determinations in cases of diabetes mellitus

In 24 cases of death in diabetic coma the peripheral venous blood showed glucose levels exceeding 3.5 mg/ml (mean value 7.76 mg/ml). In a control material of deaths of other causes the blood glucose was usually low and often zero, and all values were well below the lower limit of the diabetic concentrations. The acetone contents of the diabetic blood varied widely and were of limited diagnostic value. We conclude that glucose concentrations above 3.5 mg/ml in the peripheral blood indicate that death occurred in diabetic coma.     

Impurities, adulterants and diluents of illicit heroin in Denmark (Jutland and Funen)

The contents of impurities, adulterants and diluents in 77 samples of illicit heroin were determined by a combination of high-performance liquid chromatography and gas chromatography. The origin of each sample was characterized by calculating the content of the opium alkaloids in relation to the heroin content. The routes of distribution were compared by determination of the contents of caffeine, procaine and sugars. The results were used as a "chemical fingerprint" of each sample. The results indicate that it is difficult to prove, with certainty, that two samples are identical. However, in most cases, by determining the amounts of impurities, adulterants and diluents in heroin samples, it will be possible to ascertain whether two samples are different and, in many cases, to determine with reasonable certainty whether two samples are identical.     

Evaluation of the Postmortem Glucose and Glycogen Levels in Hepatic,Renal, Muscle,and Brain Tissues: Is It Possible to Estimate Postmortem Interval Using These Parameters?

The aim of our study was to investigate the postmortem levels of glucose and glycogen in hepatic, renal, muscle, and brain tissues and then examine the changes in those levels that could be useful for estimating postmortem interval. We established an animal model. Seventy female BALB/c albino mice were used in this study. After being sacrificed, the mice were randomly divided into six groups according to time elapsed since death (Group 1: 0 h; Group 2: 12 h; Group 3: 24 h; Group 4: 36 h; Group 5: 48 h; and Group 6: 60 h). Glucose levels were significantly different between groups for all tissues studied. Slope of the change per unit time was higher for the hepatic glucose levels. Based on these results, it is possible to estimate postmortem interval using postmortem glucose levels in hepatic tissue. Tissue‐specific assessment may contribute valuable information to postmortem interval studies.     

Macroscopic characteristics of screwdriver trauma

The main objective of the study was to determine the type of macroscopic trauma created by a flat-tipped and a cross-tipped screwdriver. The second objective was to determine if the trauma inflicted by the two screwdrivers could be macroscopically differentiated. Three tests were conducted, each by a male volunteer. Each test consisted of 12 samples of fleshed pig ribs; six were stabbed with a flat-tipped screwdriver and the remaining six with a cross-tipped screwdriver (Phillips). Each sample received 15 stab wounds during the process. The stabbings were conducted at perpendicular and oblique angles, with fabric variables being utilized. Results illustrate two main categories of macroscopic skeletal trauma, fractures, and puncture wounds. By studying the macroscopic appearance in tandem with differing trauma frequencies, these two screwdriver types can be differentiated.     

Microscopic Pattern of Bone Fractures as an Indicator of Blast Trauma: A Pilot Study

The assessment of fractures is a key issue in forensic anthropology; however, very few studies deal with the features of fractures due to explosion in comparison with other traumatic injuries. This study focuses on fractures resulting from blast trauma and two types of blunt force trauma (manual compression and running over), applied to corpses of pigs; 163 osteons were examined within forty fractures by the transmission light microscopy. Blast lesions showed a higher percentage of fracture lines through the Haversian canal, whereas in other types of trauma, the fractures went across the inner lamellae. Significant differences between samples hit by blast energy and those runover or manually compressed were observed (p < 0.05). The frequency of pattern A is significantly higher in exploded bones than in runover and compressed. Microscopic analysis of the fracture line may provide information about the type of trauma, especially for what concerns blast trauma.     

Scanning Electron Microscope Analysis of Gunshot Defects to Bone: An Underutilized Source of Information on Ballistic Trauma

Recent years have seen increasing involvement by forensic anthropologists in the interpretation of skeletal trauma. With regard to ballistic injuries, there is now a large literature detailing gross features of such trauma; however, less attention has been given to microscopic characteristics. This article presents analysis of experimentally induced gunshot trauma in animal bone (Bos taurus scapulae) using full metal jacket (FMJ), soft point (SP), and captive bolt projectiles. The results were examined using scanning electron microscopy (SEM). Additional analysis was conducted on a purported parietal gunshot lesion in a human cranial specimen. A range of features was observed in these samples suggesting that fibrolamellar bone response to projectile impact is analogous to that observed in synthetic composite laminates. The results indicate that direction of bullet travel can be discerned microscopically even when it is ambiguous on gross examination. It was also possible to distinguish SP from FMJ lesions. SEM analysis is therefore recommended as a previously underexploited tool in the analysis of ballistic trauma.     

On the detection of fingerprints by laser excited luminescence

Two surveys were carried out to determine the percentage of latent fingerprints detectable by their inherent luminescence under laser illumination. Less than one-fifth of 240 samples each containing four prints on paper gave luminescent prints. The luminescence from these was weak and only detectable under close scrutiny. Similar results were obtained for 120 samples each containing one print on glass. Forty-one of the samples showing no luminescence were sprayed, approximately one month after deposition, with a solution containing the fluorescent tagging agent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride). After treatment with NBD chloride, 37 of these samples gave luminescent prints, many of which were intensely luminescent and contained good ridge detail.