Study of cathepsin A, B and D activities in the skin wound edges. Its application to the differential diagnosis between vital and postmortem wounds

The aims of the authors in this paper has been to check the diagnostic ability of the Cathepsin A, B and D concentrations in the skin wound edges to the differential diagnosis between vital and postmortem wounds. We have studied 56 domestic pigs grouped in seven experimental series consisting of 8 animals in each, according to the time (0, 5, 15, 30 min and 1, 3 and 6 h) after the injury. The enzymatic activities were investigated following the methods by Bowen and Davison Biochem. J., 131 (1973) 417-419, Suhar and Marks J. Biochem., 101 (1979) 23-30 and by Anson (modified by Yamamoto, Eur. J. Biochem., 95 (1979) 459-467) for Cathepsin A, B and D, respectively. For the differential diagnosis between vital and postmortem wounds, our results showed that the most useful markers studied are the Cathepsin A and D activities, Cathepsin D that of the first one.     

Diagnostic ability of D-dimer in the establishment of the vitality of wounds

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.     

Localization and quantification of histamine in injured skin as parameters for the timing of wounds.

Localization and estimation of the histamine (HA) content in skin wound edges in 86 Sprague-Dawley rats and three cases of human injuries were carried out by a microfluorimetric method specific for this amine which forms a complex with o-phthalaldehyde (OPT). Distribution and density of the mast cells in the same areas were observed at the same time by toluidine blue stain. In all skin specimens with antemortem wounds, both the epidermis and upper dermis exhibit extracellular yellowish fluorescence of the HA-OPT complex. The fluorescent zone spreads in the wound edges with the lapse of time in vital injuries. The HA content increases gradually up to 30 min and then the yellow histamine fluorescence in areas 0-200 microns from the wound edge decreases. None of these features can be observed in normal skin and postmortem-injured skin. Mast cell degranulation can be demonstrated in all antemortem-injured skin. No statistical relationship exists between the number of mast cells and the HA-OPT fluorescence in either ante- or postmortem-injured groups. This study indicates that skin HA microfluorimetry by the OPT method is of practical value for distinguishing ante- from the postmortem wounds and for timing antemortem wounds.     

对纤维蛋白诊断生前伤的评价——45例枪弹创组织的MSB染色和扫描电镜对比观察

生活反应是诊断生前伤的主要根据.近二十年来,法医病理学在研究生前伤和死后伤的诊断中,采用了多种新兴技术,如扫描电镜技术、酶组织化学技术、酶标技术、电泳技术等,促使生前伤的诊断水平有明显提高.伤后存活1小时死亡者,大部分皆可获得明确诊断;立即死亡或在濒死期形成的损伤,由于生活反应甚微,或受腐败等     

A study on the vital reaction in wounded skin: simultaneous determination of histamine and polyamines in injured rat skin by high-performance liquid chromatography

This paper describes a modified method of simultaneous quantitative determination of histamine and polyamines (putrescine, cadaverine, spermidine and spermine) in incised skin wounds of rats using high-performance liquid chromatography (HPLC) with a strong-cation exchange column, shim-pack ISC-05/S0504P, employing a linear gradient of 0.7-2.5 M sodium chloride. There was scarcely any change in the histamine content of the vital wounds throughout the one week experimental period, although content decreased slightly in 1-h-old wounds. On the other hand, concentrations of putrescine and spermidine increased suddenly 12 h after wounding, and spermine content increased slightly in 4-day-old wounds. There was no post-mortem change in the histamine, spermidine and spermine contents of vital wounds allowed to stand at 25 degrees C up to 24 h after death. Based on these results, this HPLC method may be useful for the determination of wound age during the period of healing.     

Immunohistochemical study of fibronectin for postmortem diagnosis of early myocardial infarction

The postmortem diagnosis of early myocardial infarction has been a puzzling problem in forensic practice. In the present study, an immunohistochemical study of fibronectin (FN) was performed for the first time on 34 autopsy hearts to determine early myocardial infarction with streptavidin/biotin/peroxidase technique. Five cases of definite myocardial infarction showed positive FN staining of cardiomyocytes; of 18 cases where early myocardial infarction was suspected, positive FN staining of cardiomyocytes was found in 15 cases, but no such staining was seen in 11 non-cardiac death controls. The results led to the conclusion that positive FN staining in cardiomyocytes is a reliable marker of acute myocardial infarction and could be used as a new, sensitive method for the postmortem diagnosis of early myocardial infarction. It is worth noting that all cases in this study were autopsied between 8 h and 4 days after death and 5 cases had been fixed in 10% formalin for over 10 years. FN immunohistochemistry still gave satisfactory results in those cases. It seemed that FN was not affected by postmortem autolysis and formalin-fixation and could be used in routine forensic practice, especially for retrospective analysis of cases.     

Biochemical reconstruction of three cases of death — Results of international cooperation

Biochemical serotonin and histamine determinations were applied to the reconstruction of three suspected homicide cases. To distinguish between ante-mortem and post-mortem wounds and to time the ante-mortem injuries the concentrations of free histamine and serotonin in the wound samples and in the control samples from neighbouring intact skin were examined. The results of these biochemical determinations allowed a reconstruction of the events and one of the three cases was shown to be suicide instead of homicide.These methods can be used at least during the first 4 – 5 days after death and some-times even longer. This allows for international cooperation when investigating and reconstructing complicated cases of death.     

损伤皮肤中组胺荧光显微定位定量与损伤时间推断的实验研究

本文应用显微荧光分光光度计法研究大白鼠皮肤创缘组织中组胺的分布和含量,并用甲苯胺蓝法观察创缘肥大细胞形态和数量的变化。结果发现,创缘真皮乳头层出现扩散的细胞外黄色荧光和肥大细胞脱颗粒现象为生前伤的重要特点;组胺荧光分布范围及增多的程度与损伤时间密切相关。从而为损伤时间推断提供了一种新的方法。     

The Effects of Sharp‐Force Thoracic Trauma on the Rate and Pattern of Decomposition

Given the multiple factors that may alter the rate of decomposition, one of the more difficult tasks that anthropologists and pathologists face is determining of the postmortem interval (PMI). While trauma has been widely accepted as being among those factors, recent published studies have contradicted this premise. However, major issues exist with these studies. Consequently, this study was designed to simulate a more realistic setting in an effort to settle this discrepancy in the literature. This study utilized eight porcine remains; three of which received incised wounds penetrating the thoracic cavity, three others received nonpenetrating wounds, and two were designated nontrauma controls. Measurements included evaluating the total body score (TBS) on temporal and accumulated degree days (ADD). Using a two‐way repeated measures ANOVA, the results of this study found that while trauma does have an influence in the pattern of decomposition, it does not influence the rate of decomposition.     

Determining the Effectiveness of Noncontact Three-Dimensional Surface Scanning for the Assessment of Open Injuries

Noncontact three-dimensional (3D) surface scanning methods are used within forensic medicine to record traumas and other related findings. A structured light scanning technique is one of these methods and the most suitable for the forensic field. An assessment of the efficiency of different structured light scanners with forensic injuries is essential to validate this technique for wound documentation. The purpose of this study was to evaluate the capability of the HP structured light scanner Pro S3 for digitizing open injuries having complicated areas and depths. Fifteen simulated injuries on mannequins were scanned and assessed. Comparisons between 3D and direct wound measurements were made. The results showed that the technique was able to create detailed 3D results of the extensive injuries. Statistical significance tests revealed no difference between the two measurement methods. Because the scanner is applicable for routine work, it should be considered to confirm the same results on real cadavers and actual wounds.     

Postmortem stability of some markers of intra-vital wounds

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.     

Forensic veterinary radiology: Ballistic-radiological 3D computertomographic reconstruction of an illegal lynx shooting in Switzerland

The lynx, which was reintroduced to Switzerland after being exterminated at the beginning of the 20th century, is protected by Swiss law. However, poaching occurs from time to time, which makes criminal investigations necessary. In the presented case, an illegally shot lynx was examined by conventional plane radiography and three-dimensional multislice computertomography (3D MSCT), of which the latter yielded superior results with respect to documentation and reconstruction of the inflicted gunshot wounds. We believe that 3D MSCT, already described in human forensic-pathological cases, is also a suitable and promising new technique for veterinary pathology.     

Breaking point? An analysis of fatal stab wounds to the torso in Ireland between 2011 and 2018, examining the extent of rib fractures

This study examines the pattern of rib injuries occurring in cases of fatal torso stab wounds in Ireland between 2011 and 2018. It has been suggested by previous studies that rib fractures are not commonly sustained in stab wounds to the torso. We wanted to ascertain whether this was the case, as our data suggested that rib fractures were frequent, and where a rib is fractured there is a higher chance of organ injury and death, making this an important area of study. One hundred and forty seven cases of fatal stab wounds from an eight-year period were retrospectively reviewed. Fatal stab wounds to other body areas, were excluded; leaving a total of 104 cases with stab wounds to the torso. We found that 69.2% of cases had rib injuries, a figure significantly higher than previously reported. Our data suggests that stab wounds to the torso often fracture ribs, putting the underlying organs at increased risk of injury and perhaps contributing to fatality. The amount of force needed to cause a rib fracture can be difficult to quantify and indeed from the high percentage of rib fractures sustained in our data it appears that the ribs may be fractured regardless of the amount of force used; this is borne out by the finding that self-inflicted injuries also caused rib fractures. Our study shows that other factors, such as anatomical positioning and wound depth may have a greater bearing than force in terms of whether a rib fracture is sustained.     

Expression of fibronectin suicidal in gunshot wounds

Fibronectin has been proposed as the reliable marker of wound vitality and potent tool for age estimation in cases where survival time is at least few minutes. Cases of suicidal gunshot wounds were divided into two groups according to projectile energy and damage of the vital structures. Immunohistochemical reaction on fibronectin was evaluated with semiquantitative scale, which included sum of intensity and widespread of the reaction. Mann–Whitney test has shown statistical significant difference between groups (P ≤ 0.001).     

Initial Study for the Determination of the Sequence of Intersecting Lines between Gel Pens and Seals by Optical Coherence Tomography*

Determining the sequence of intersecting lines is a significant issue in the forensic document examination that can reveal the fraud or distinguish between different allegations. Optical coherence tomography (OCT) is a high-resolution cross-sectional imaging technique that has been introduced into forensic science field recently. The potential of OCT as a novel method to determine the sequence of intersecting lines was examined for the first time. In this study, a spectral-domain OCT system with a center wavelength of 900 nm was employed to perform nondestructive examination on determining the sequence of 18 heterogeneous intersecting line samples produced using three types of gel pens and three brands of stamp pad ink seals. Two-dimensional (2D) cross-sectional, and three-dimensional (3D) volumetric images of the intersecting lines were obtained by the OCT system. Several features were noted and analyzed to successfully determine the sequence of all the 18 samples. Blind tests were also conducted to demonstrate the effectiveness of OCT technique. The results illustrate that OCT technology can provide an effective and accurate method for sequencing intersecting lines of gel pen ink and seal ink, which may complement the conventional methods used in the examination of questioned documents.     

A Molecular Method to Correlate Bloodstains with Wound Site for Crime Scene Reconstruction

Bloodstain pattern analysis to determine the wound‐of‐origin of bloodstains is problematic with nonspecific patterns. In this proof‐of‐concept study, the authors examined a molecular approach to correlate bloodstains with injuries using the rat as a model. Specifically, investigations were conducted on the rat brain marker, rno‐miR‐124‐3p, with the QIAGEN miScript System and real‐time PCR analysis. Rno‐miR‐124‐3p was detected in brain homogenates diluted 100,000 times; in 3‐week‐old, room temperature stored, simulated brain–blood stains; and in bloodstains from head gunshot wounds collected with swabs and subsequently frozen for 9–18 months; however, rno‐miR‐124‐3p was not detected in whole blood. Proof‐of‐principle was demonstrated by the ability to distinguish bloodstains from a gunshot wound to the head versus bloodstains from a gunshot wound to the chest, by the testing of otherwise identical bloodstains from the two patterns for the presence of the marker. The results suggest a viable approach to a longstanding problem in casework.     

Hesitation Wounds and Sharp Force Injuries in Forensic Pathology and Psychiatry: Multidisciplinary Review of the Literature and Study of Two Cases

The aim of this paper was to showcase the significant diagnostic value of hesitation wounds in terms of forensic, psychiatric, and medicolegal interest. A number of studies were reviewed to update and summarize the relevant literature on the incidence, distribution, character, and function of hesitation wounds as well as the sociodemographic variables and psychopathology of the inducers. This study also investigates their importance as a forensic criterion in the distinction between suicide and homicide as well as a psychiatric diagnostic tool in suicide prevention. In addition, the paper reports two new cases. Results conclude that there is equal incidence, but different distribution of hesitation wounds between genders. Furthermore, the low dispersion of hesitation wounds contrasts with the high dispersion of defense wounds. The inducers' psychopathology lies principally in Axis I disorders. Finally, there is a comprehensive analysis of non‐suicidal self‐injury and the role of self‐wounding in suicide prevention.     

Paternity testing by the detection of D1S80 VNTR using fluorescence image analyzer (Dualcolour system)

An improved method for DNA polymorphism typing of D1S80 VNTR locus and its application to paternity testing are described. For accurate estimation of the length of polymorphic DNA fragments, the size marker was labeled with fluorescence different from that of PCR primers, and co-electrophoresed as an internal standard. The dualcolour system of fluorescence image analyzer was used to detect the fragments and determine their size. This internal marker method could successfully overcome the problems of band pattern distortion and tailing, besides it allows easy and accurate interpretation of the DNA types. Our results indicate that the internal marker method is much more accurate than the method of using size marker in gel, even with the presence of distortion or tailing of the band patterns. Family studies applying this method showed complete agreement between the observed and predicated types.