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Purpose: The main objective of this study was to test, for the first time, a highly sensitive cardiac troponin T (cTnThs) assay in postmortem serum and pericardial fluid and to evaluate cardiac troponin T (cTnT) levels and their stability after death at different postmortem intervals, in an attempt to determine the viability of the cTnThs assay in the postmortem diagnosis of the cause of death., Methods: cTnT levels were determined in serum and pericardial fluid samples taken from 58 cadavers at known postmortem intervals, whose causes of death were categorized into the following groups: (1) sudden cardiac deaths, (2) multiple trauma, (3) mechanical asphyxia, and (4) other natural deaths. cTnT was determined by inmunoassay, using the Troponin T highly sensitive STAT assay (Roche(®))., Results: Average cTnT levels measured by a highly sensitive assay in postmortem serum were markedly higher than clinical serum levels. Moreover, similar results, higher cTnT levels in postmortem pericardial fluid, were obtained when compared to levels found in pericardial fluid taken from two living patients during coronary artery bypass surgery. cTnT levels in both postmortem fluids remained stable for up to 34 h after death. No differences in cTnT levels in either postmortem fluid by sex and age were detected. Levels of cTnT found in pericardial fluid in the other natural deaths group were significantly lower than the cTnT levels found in that postmortem fluid from any of the other causes of death groups., Conclusion: It is therefore reasonable to conclude that determination of cTnT by a highly sensitive assay in pericardial fluid can provide forensic pathologists with a complementary test to the diagnosis of cause of death.

Analyses of gene expression of ischemic myocardial injury and repair related proteins has been carried out for the first time in samples from five specific sites of the myocardium, pericardial fluid and blood from thirty cadavers in relation to post-mortem interval (PMI). RNA integrity was evaluated by RNA integrity number (RIN), with values ranging from 6.57 to 8.11; sufficiently high levels of integrity to permit further gene amplification. No significant correlations between RIN and PMI in any samples were detected. Prior to target gene expression analysis, a normalization strategy was carried out to assess candidate reference gene stability, involving the analysis and comparison of four common housekeeping genes (Glyceraldehide-3-phosphate dehydrogenase, beta-actin, TATA box binding protein and Cyclophilin A). Gene expression of cardiac troponin I (TNNI3), myosin light chain 3 (MYL3), matrix metalloprotease 9 (MMP9), transforming growth factor beta 1 (TGFB1), and vascular endothelial growth factor A (VEGFA) in myocardial zones and body fluids were subsequently studied by real-time quantitative PCR. Expression levels of all the proteins studied in cardiac zone samples were similar. No statistical differences for expression were detected among proteins taken from any myocardial area. No significant differences were detected for TNNI3 and TGFB1 gene expressions when compared with samples at or under 12h-PMI or over 12h-PMI. However, differences in MYL3, MMP9, and VEGFA gene expression in body fluids were found at PMI periods of over 12h. These interesting results may contribute to the refinement of current knowledge regarding cardiac metabolism and improve understanding of the underlying mechanisms involved in myocardium ischemia and its repair., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The potential of skin measurements of D-dimer (DD), a specific derivative of crosslinked fibrin, for the diagnosis of the vital origin of wounds was investigated in 67 human vital skin wounds and homolateral control pieces of skin from 53 autopsies performed at the Institute of Legal Medicine of Coimbra (Portugal) during 1992. Our results demonstrate that DD levels for the incised vital skin wounds (n = 50) were much higher on average than their controls (P < 0.01) which were actually incised injuries produced postmortem at the autopsy. We did not find any statistical difference in average DD levels among the remaining injuries (seven abrasions and ten contusions) in comparison to their respective controls. DD may be used, with certain limitations, as a marker of the vitality of incised wounds. The authors believe this is the first time this marker has been applied in forensic pathology. It is an example of an easy and quick application technique, with the added advantage of low cost.

We have studied the viability of PGF2a as a vitality marker in skin wounds. Incised vital skin wounds and homolateral control pieces of skin were obtained from 20 autopsies performed at the Institute of Legal Medicine of Coimbra University (Portugal). We have also studied 10 fresh skin samples from the Department of Dermatology of the University Hospital (Granada). Our results show that PGF2a is not suitable for the diagnosis of the vitality of wounds because of its irregular behaviour.

This paper shows the results obtained by studying the lysosomal enzyme Cathepsin D as a potential marker for the vitality of wounds in human specimens. We have analyzed 53 samples using enzymological and histological techniques. Our results show the ability of Cathepsin D to establish the vital origin of wounds inflicted 5 minutes or less before death, where the specific activity of cathepsin D reached 0.055 units at the wound edge and 0.01 units in their respective controls (P < 0.001). As previously demonstrated in an experimental series, Cathepsin D seems to be a very useful marker of high forensic interest in especially difficult cases. Further studies are in progress to check the influence of different factors such as drugs intake and clinical conditions on Cathepsin D activity.

The postmortem stability of the main phospholipids of lung surfactant-phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI), phosphatidyl serine (PS) and sphingomyelin (S) in three different deaths; one caused by fresh-water drowning, one by salt-water drowning, and one from a sodium-pentobarbital overdose has been studied. The drug overdose was considered the control because there was no surfactant involvement. The results show the stability of these kinds of lipids in the first 24 h, with a progressive decrease from 48 h on until 96 h, with a significant correlation to the time of P less than 0.01 in most cases.

We have studied the diagnostic value of several markers of the intra-vital nature of wounds - cathepsin D (EC 3.4.23.5) and ions (Ca, Mg, Cu, Zn and Fe) - after the influence of putrefaction. For this purpose, we have inflicted vital wounds to six pigs, which were killed 20 min later. Ten minutes after death, wounds were excised with 5-6 cm of skin around the incision and maintained at three different temperatures (4, 18 and 28.5 +/- 13.4 degrees C). After varying periods of postmortem interval from 0 to 48 h, aliquots of each wound were taken and analyzed with atomic absorption spectrophotometry for ions and with UV-spectrophotometry for cathepsin D. Our results demonstrate that ions conserve their diagnostic ability to differentiate vital from postmortem wounds after the influence of putrefaction. Nevertheless, cathepsin D does not show this ability in these experimental conditions.

The activities of the rate-limiting enzymes of glycolytic pathway were measured in various areas of rat brains kept at a temperature of +25 degrees C for various intervals after death by cervical dislocation. Hexokinase shows a substantial decline in activity over a period of 24 h, reaching 41%, 57%, 44%, and 51% of the controls in cerebellum, medulla oblongata and pons, cerebral cortex, and diencephalon, respectively. In the same areas the phosphofructokinase reached 28%, 61%, 60%, and 40% of the zero-time activity, respectively. Lactate dehydrogenase behaves differently in the four areas, with an increase in cerebral cortex and diencephalon and a decrease in cerebellum and medulla oblongata and pons. Pyruvate kinase activity was quite stable over the 24 h period studied. Therefore, the activities of hexokinase and phosphofructokinase in brain tissue were of little value for diagnosis of the time of death.

The postmortem stability of the rat Atrial Natriuretic Peptide (ANP) has been studied as a necessary and previous step to be applied in the forensic field as a postmortem marker. This peptide--whose extreme sensitivity to slight changes in blood volume is well known--could have great importance in thanatochemistry to establish a correct diagnosis when macroscopical observations and classical parameters are not conclusive or cannot be employed. The results show high stability in atrial tissue, where values are similar from 0 hours (108.99 pm/mg) to 8 hours (109.41 pm/mg) and decrease uniformly until 15 pm/mg) at 32 hours, time of our last determination. Blood ANP showed similar stability from 0 h (105.43 pg/ml) to 8 h (106.62 pg/ml).

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.