Detection of fibrocytes in human skin wounds and its application for wound age determination

Fibrocytes, a newly identified cell type, are bone marrow-derived mesenchymal progenitors that coexpress hematopoietic cell antigens and fibroblast products. In this study, a double-color immunofluorescence analysis was carried out using anti-CD45 and anti-collagen type I antibodies to examine the time-dependent appearance of fibrocytes, using 53 human skin wounds with different wound ages (group I, 0–3 days; group II, 4–7 days; group III, 9–14 days; and group IV, 17–21 days). In wound specimens with an age of less than 3 days, CD45⁺/collagen type I⁺ fibrocytes were not detected. The fibrocytes were initially observed in wounds aged 4 days, and their number increased in lesions with advances in wound age. In a semiquantitative morphometrical analysis, the average number of fibrocytes was highest in the wounds of group III. These findings imply that human skin wounds containing fibrocytes are at least 4 days old. Moreover, a fibrocyte number of over 10 indicates a wound age between 9 and 14 days (i.e., group III). Based on the average number of fibrocytes in each group, a fibrocyte number of over 15 more strongly suggests a wound age of 9–14 days. Together, our observations indicate the participation of fibrocytes in wound healing of human skin inducing the accumulation of extracellular matrix components, and therefore, detection of fibrocytes could be a useful marker for wound age determination.     

Forensic application of VEGF expression to skin wound age determination

An immunohistochemical study combined with morphometry was carried out to examine the time-dependent expression of vascular endothelial growth factor (VEGF) using 53 human skin wounds with different wound ages (groups I: 0–12 h, II: 1–4 days, III: 7–14 days and IV: 17–21 days). In the human wound specimens aged 4–12 h, neutrophils recruited at the wound showed no positive signals for VEGF. With an increase in wound ages of 7 days, granulation tissue and angiogenesis were observed, with the migration of macrophages and fibroblasts of which the cytoplasm expressed VEGF-positive reactions. Morphometrically, the average VEGF-positive ratio was highest in group III, followed by that of group IV. In groups III and IV, 13 out of 26 wound samples had VEGF-positive ratios of more than 50%. However, all of the wound samples in groups I and II showed VEGF-positive ratios of less than 50%. With regard to the practical applicability and forensic validity, these observations suggest that a VEGF-positive ratio of more than 50% possibly indicates a wound age of 7 days or more.     

Immunohistochemical detection of MMP-2 and MMP-9 in a stasis-induced deep vein thrombosis model and its application to thrombus age estimation

We immunohistochemically examined the expression of matrix metalloproteinase (MMP)-2 and MMP-9 using venous thrombi developed by ligation of the inferior vena cava (IVC) in mice. Both MMP-2- and MMP-9- positive cells could be detected in the whole course of thrombus formation after IVC ligation. Morphometrically, their number was greatest 14 days after IVC ligation and thereafter, gradually decreased at 21 days. The number of MMP-9-positive cells was significantly higher than that of MMP-2-positive cells at 1 to 7 days. The average ratio of MMP-9 to MMP-2 (MMP-9/MMP-2 ratio) was >2.0 in all thrombus samples at 1–5 days. After 7 days, the MMP-9/MMP-2 ratio was less than 2.0. These observations implied that an MMP-9/MMP-2 ratio markedly exceeding 2.0 strongly indicates an age of 5 days or less. Furthermore, an MMP-9/MMP-2 ratio of <2.0 probably indicates an age of more than 7 days. The present study demonstrated that the immunohistochemical detection of intrathrombotic MMP-2 and MMP-9 was suitable to estimate the age of venous thrombi.     

Expression of oxygen-regulated protein 150 (ORP150) in skin wound healing and its application for wound age determination

Immunohistochemical study combined with morphometry was carried out to examine the expression of oxygen-regulated protein 150 (ORP150) using 58 human skin wounds of different ages (group I, 0-12 h; II, 1-5 days; III, 7-14 days; and IV, 17-21 days). In human wound specimens aged 4-12 h, neutrophils recruited at the wound showed no positive signals for ORP150. With the increase in wound age of >/=7 days, granulation tissue and angiogenesis were observed, with the migration of macrophages and fibroblasts with ORP150-positive reactions. In semi-quantitative analysis, the average of ORP150-positive ratios in group III was highest. In group III, all samples had an ORP150-positive ratio of >40%, and 17 samples showed >50%. In group IV, three out of ten samples showed a positive ratio of 40-45% and the remaining seven cases less than 40%. Collectively, with regard to the practical applicability with forensic safety, these observations suggest that an ORP150-positive ratio of >50% strongly indicates a wound age of 7-14 days.     

Immunohistochemical detection of uPA,tPA, and PAI-1 in a stasis-induced deep vein thrombosis model and its application to thrombus age estimation

We immunohistochemically examined the expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasminogen-activator inhibitor type-1 (PAI-1) using venous thrombi developed by ligation of the inferior vena cava (IVC) in mice. The uPA-, tPA- and PAI-1-positive cells could be firstly detected 5, 7, and 3 days, respectively, after IVC ligation. Morphometrically, the number of PAI-1-positive cells was significantly higher than those of uPA- and tPA-positive cells at later than 7 days. In all of the thrombus samples aged 10–21 days, the uPA/PAI-1 and tPA/PAI-1 ratios were >0.1 and >0.2, respectively. In contrast, all of the thrombus samples aged 1–7 days had uPA/PAI-1 of <0.1 and tPA/PAI-1 ratios of <0.2. These findings implied that uPA/PAI-1 of >0.1 and tPA/PAI-1 of >0.2 indicated an age of 10 days or more. Moreover, in four of five samples aged 10 days, uPA/PAI-1 ratios were <0.3, and the remaining one had uPA/PAI-1 of 0.32. All thrombi aged 14–21 days showed values greater than 0.3. Thus, uPA/PAI-1 ratios, markedly exceeding 0.3, strongly indicated an age of more than 14 days. The present study demonstrated that the immunohistochemical detection of uPA, tPA, and PAI-1 was suitable to estimate the age of venous thrombi.     

The application of autophagy to thrombus age estimation in murine deep vein thrombosis model

International Journal of Legal Medicine - We immunohistochemically examined the intrathrombotic dynamics of autophagy during thrombogenesis using murine deep vein thrombosis (DVT) models. To...     

Detection of intrathrombotic endothelial progenitor cells and its application to thrombus age estimation in a murine deep vein thrombosis model

International Journal of Legal Medicine - Endothelial progenitor cells (EPCs), a newly identified cell type, are bone marrow-derived progenitor cells that co-express stem cell markers and Flk-1...     

Immunohistochemical detection of intrathrombotic macrophage-derived cytokines and its application to thrombus age estimation in murine deep vein thrombosis model

We immunohistochemically examined the expression of IFNγ, TNFα, and TNF receptor p55 (TNF-Rp55) in a stasis-induced venous thrombus murine model. IFNγ⁺, TNFα⁺, and TNF-Rp55⁺ cells could be first detected 7, 3, and 3 days after inferior vena cava (IVC) ligation. Thereafter, the numbers of these positive cells increased with time after IVC ligation. Double-color immunofluorescence analyses demonstrated that each molecule was expressed by intrathrombotic macrophages. In all samples with postligation intervals of 7 days or less, IFNγ/macrophage ratio (IFNγ/M?), TNFα/macrophage ratio (TNFα/M?), and TNF-Rp55/macrophage ratio (TNF-Rp55/M?) were <0.2, <0.2, and <0.4, respectively. In contrast, IFNγ/M? and TNFα/M? were greater than 0.2 and 0.3 at ≥10 days after the IVC ligation, respectively. These observations suggested that IFNγ/M? ratios of >0.2 and TNFα/M? ratios of >0.3 indicated thrombus age of ≥10 days. Moreover, TNF-Rp55/M? ratios of >0.7 strongly suggested thrombus age of >14 days. The present study demonstrated that the immunohistochemical detection of IFNγ, TNFα, and TNF-Rp55 was suitable to estimate the age of venous thrombi. However, the variation in TNF-Rp55/M? ratios is wider than those of the other two markers. Thus, IFNγ and TNFα could be more practical markers.     

Immunohistochemical analysis of markers for different macrophage phenotypes and their use for a forensic wound age estimation

A total of 117 vital skin wounds (post infliction intervals between a few seconds and 7 months), 20 postmortem wounds and skin specimens with beginning or advanced signs of putrefaction were investigated. Different markers for macrophage maturation (27 E 10, RM 3/1, 25 F 9, G 16/1) were analyzed by immunohistochemistry. The early stage inflammation marker 27 E 10 stained macrophages, but also monocytes and neutrophilic granulocytes localized in blood vessels or bleeding induced postmortem and therefore provided no further information for a forensic wound age estimation in comparison to the routine histological detection of macrophages. The antigens recognized by the RM 3/1- (intermediate stage inflammation marker) and 25 F 9-antibodies (late stage inflammation marker) were expressed exclusively by histiocytes and inflammatory cells that had migrated from the blood vessels as part of the acute inflammatory response associated with an intravital reaction. The morphometrical analysis revealed positive results (defined as at least a two-fold increase in number in 2 or more microscope fields when compared to the maximum value of histiocytes found in uninjured skin) for the RM 3/1- or 25 F 9-antibody earliest in wounds aged 7 or 11 days, respectively. Similarly to the 25 F 9-antibody, the chronic stage inflammation marker (G 16/1) reacted with a macrophage subpopulation first detectable 12 days after wounding but showed positive results in a comparably reduced percentage of cases. On the other hand, this marker did not stain a relevant number of resident macrophages thus facilitating the evaluation of the specimens. The markers 27 E 10, RM 3/1 and 25 F 9 are also useful for the evaluation of slightly - even though the staining intensity was considerably reduced - but not advanced putrefied skin. Therefore, the immunohistochemical analysis of the corresponding antigens can possibly contribute to an age estimation of wounds with advanced post infliction intervals obtained from corpses with longer - but limited - postmortem intervals.

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Zusammenfassung Insgesamt wurden 117 vitale Hautwunden (Überlebenszeit wenige Sekunden bis 7 Monate), 20 postmortal gesetzte Verletzungen sowie Haut mit leichten bzw. fortgeschrittenen Fäulnisveränderungen untersucht und verschiedene Marker der Makrophagen-Differenzierung (27 E 10, RM 3/l, 25 F 9 und G 16/1) analysiert. Der early stage inflammation marker 27 E 10 färbte neben Makrophagen auch Monozyten und neutrophile Granulozyten, die innerhalb von Blutgefäßen bzw. in postmortal gesetzten Blutungen lokalisiert waren und liefert somit keine Informationen zum Wundalter, die über die Möglichkeiten des Routine-histologischen Nachweises von Makrophagen hinausgingen. Die von den Antikörpern RM 3/1 (intermediate stage inflammation marker) und 25 F 9 (late stage inflammation marker) erkannten Antigene wurden ausschließlich von Histiozyten und reaktiv eingewanderten Makrophagen exprimiert. Die morphometrische Analyse ergab positive Ergebnisse (definiert als ein mindestens zweifacher Anstieg der Zellzahl in zwei oder mehr Gesichtsfeldern verglichen mit der maximal feststellbaren Zahl an Histiozyten in unverletzter Haut) bei Verwendung der Antikörper RM 3/1 bzw. 25 F 9 frühestens 7 bzw. 11 Tage nach Wundsetzung. Ab 12 Tagen Wundalter reagierte der chronic stage inflammation marker G 16/1 erstmals positiv. Das Antigen ließ sich insgesamt allerdings in einem geringeren Prozentsatz der untersuchten Wunden darstellen. Vorteilhaft ist jedoch das Fehlen einer relevanten Expression durch Histiozyten, wodurch die Auswertung der Präparate erleichtert wird. Die entsprechenden Antigene lassen sich zudem in leicht - wenn auch in einer deutlich geringeren Färbeintensität -, aber nicht forgeschritten fäulnisveränderter Haut nachweisen, so daß deren immunhistochemische Darstellung gegebensfalls auch zur Beurteilung von länger überlebten Verletzungen an Leichen mit etwas fortgeschrittener Liegezeit herangezogen werden kann.

     

Immunohistochemical detection of chemokines in human skin wounds and its application to wound age determination

Immunohistochemical studies on the time-dependent expression of the chemokines such as interleukin (IL)-8, monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-1α were performed on 50 human skin wounds with different wound ages (group I 0–12 h, group II 1–4 days, group III 7–14 days and group IV 17–21 days). In the wound specimens with wound ages between 4 and 12 h, neutrophils mainly showed positive reactions for IL-8, MCP-1 and MIP-1α. With increasing wound ages, macrophages and fibroblasts were positively stained with anti-IL-8, MIP-1α and MCP-1 antibodies. Morphometrically, there was a similar distribution in the positive ratios of the inflammatory cells among IL-8, MCP-1 and MIP-1α. The positive ratios of each chemokine were very low in group I and a considerable increase of the positive ratios in each chemokine was observed in group II (mean ± standard error IL-8: 59.8 ± 2.1%, MCP-1: 42.4 ± 3.1% and MIP-1α: 50.4 ± 3.7%). Although the positive ratios for each chemokine gradually decreased according to the wound age, the mean positive ratios in groups III and IV were significantly higher than those in group I. From the forensic aspect, these chemokines are considered useful markers for wound age determination. Thus, ratios of > 50% for IL-8, > 30% for MCP-1 or > 40% for MIP-1α indicate a wound age of at least 1 day. Moreover, the combined investigation of these three chemokines can make wound age determination more objective and accurate. Received: 30 May 2001 / Accepted: 14 August 2001     

Assessment of dental age estimation methods applied to Brazilian children: a systematic review and meta-analysis

Objectives:To review the scientific literature of studies on dental age estimation methods applied to Brazilian children.Methods:A systematic literature review was designed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and registered in PROSPERO (CRD42020136170). Six scientific databases were used as primary search sources (PubMed, Scopus, LILACS, SciELO, Embase and Web of Science) and two databases (Open Grey and Open Thesis) were searched to partially select the “grey literature.” Only cross-sectional studies were included. The risk of bias was assessed by means of Joanna Briggs Institute Critical Appraisal Tools for Systematic Reviews. The standardized mean difference (SMD) between the estimated dental and chronological ages was meta-analysed via random effects model.Results:The search resulted in 2,527 studies, from which 13 met the eligibility criteria. Out of the eligible studies, 76.92% had low risk of bias and high methodological quality. Ten studies provided proper information to be included in the meta-analysis.The methods and their SMD between estimated and chronological ages were: Willems’=0.05, Lilequist and Lundberg’s = −0.11, Nolla’s = 0.22, Mornstad’s = 0.27, Cameriere’s = −0.31, Demirjian’s = 0.74 and Haavikko’s = −0.87.Conclusion:Although originally trained in populations worldwide, most of the international methods for radiographical dental age estimation had optimal performance in Brazilian children.     

可回收腔静脉滤器在下肢深静脉血栓导管抽吸去栓术治疗中的应用

目的：探讨国产可回收腔静脉滤器在急性下肢深静脉血栓形成经皮导管抽吸去栓术中的应用价值。方法：29例左侧急性下肢深静脉血栓形成,预先置入可回收腔静脉滤器,随后行导管抽吸去栓,最后对左侧髂总静脉行血管成形术。结果：29例中27例回收,19例截获大块血栓,所有病例均未发生肺栓塞及与滤器相关的并发症。结论：行经皮导管抽吸去栓术预先置入可回收性滤器,是一种安全有效的必要措施。     

Combining dental and skeletal evidence in age classification: Pilot study in a sample of Italian sub-adults

BackgroundDental and skeletal maturation have proved to be reliable evidence for estimating age of children and prior studies and internationally accredited guidelines recommend to evaluate both evidence in the same subject to reduce error in age prediction. Nevertheless the ethical and legal justification of procedures that imply a double exposition of children stands as a relevant issue. This study aims to evaluate the accuracy of age estimation provided by a combination of skeletal and dental methods applied in the same sample of children.Materials and methodsThe sample consisted of 274 orthopantomographies and left hand-wrist X-rays of Italian children, (aged between 6 and 17 years) taken on the same day. Greulich and Pyle’s (GP), Tanner-Whitehouse’s version 3 (TW3) and Willems’ (W) and the Demirjian’s (D) methods were respectively applied for estimating skeletal and dental age. A combination of skeletal and dental age estimates through Linear Discriminant Analysis (LDA) is proposed to obtain a classifier respect to an age threshold.ResultsThe combination of D and TW3 obtained an improvement of accuracy in classifying female subjects respect to the 12 years threshold respect to the original methods (from about 77% using either original methods to 83.3% combining TW3 + D) as well as a consistent reduction of false positives rate (from 17% to 21% for original methods to 5.6% with TW3 + D). For males the LDA classifier (based on TW3 and W) enable a small improvement in accuracy, whilst the decreasing of false positives was as noticeable as for females (from 17.6 to 14.1% for original methods to 6.2% combining TW3 + W).ConclusionsAlthough the study is influenced by the limited size and the uneven age distribution of the sample, the present findings support the conclusion that age assessment procedures based on both dental and skeletal age estimation can improve the accuracy and reduce the occurrence of false positives.     

Evaluation of the medial clavicular epiphysis according to the Schmeling and Kellinghaus method in living individuals: A retrospective CT study

The evaluation of the medial clavicular epiphysis via CT plays an important role in the determination of age, particularly the 19th and 22nd ages. Several authors have recommended the use of the Schmeling and Kellinghaus methods in conjunction in the evaluation of the medial clavicular epiphysis. The aim of this retrospective study was to evaluate thin section CT scan images of the medial clavicular epiphysis according to the Schmeling and Kellinghaus method, and to discuss the obtained data in the light of the literature. The thoracic CT scan images (0.6 mm section thickness) of 601 patients (202 female and 399 male) aged between 10 and 35 years obtained by 16-detector CT were evaluated by two examiners. The stage 2 was seen between 13 and 23 years of age; stage 3 was seen between 16 and 27 years of age. However, 100% of the female cases with stage 3c were ⩾18 years of age, and 100% of the male cases with stage 3c were ⩾19 years of age. Stage 4 was first observed at 20 years of age in both sexes, and stage 5 was first observed at 25 years of age in both sexes. We believe that stage 3c may be used, particularly in the determination 18-year age limit for both sexes. The outcomes of our study are consistent with those of our previous study and other studies in the literature, which is important for the confirmation of the reliability of the method.     

Performance of wells score to predict deep vein thrombosis and pulmonary embolism in endurance athletes

Introduction: There are an increasing number of reports describing deep vein thrombosis (DVT) and/or pulmonary embolism (PE) in otherwise healthy endurance athletes. The Wells score is the most commonly used clinical prediction rule to diagnose DVT/PE in clinical populations. However, the Wells score may have limited utility for recognition of DVT/PE in athletes, contributing to missed or delayed diagnosis.

Objective: We performed an analysis of the ability of the Wells score to identify DVT/PE events in athletes through a review of published case reports.

Methods: A systematic search of the literature yielded 11 case reports.

Results: The Wells score had a 100% failure rate in identifying athletes with DVT (0/6) and PE (0/5), resulting in a delayed diagnosis for DVT of 20 ± 14 days. Retrospectively removing ‘differential diagnosis’ from the clinical prediction rule for DVT changed the Wells score median from 0 (range: ?1 to 0) to 2 (range: 1 to 2); the threshold for predicting DVT as ‘likely’. There were limited clinical characteristics captured in the Wells score for PE that were applicable to athletes, highlighting the need for reappraisal. Although the Wells score failed to accurately triage athletes with known DVT and/or PE, the addition of a D-dimer value (mean: 1566 ± 758ng/dL) to the Wells score correctly identified 9/9 athletes.

Conclusions: The Wells score had a 100% failure rate for triaging athletes with known DVT/PE. When performed, D-dimer adequately facilitated the additional diagnostic testing required for a timely diagnosis of DVT/PE in athletes. Improving awareness of an atypical presentation of thrombotic events in athletes may reduce the widespread underestimation of DVT/PE among athletes and facilitate the additional testing required for a timely diagnosis.     

Diagnostic yield and characteristic features in a series of decomposed bodies subject to coronial autopsy

A study was undertaken of 629 cases autopsied at Forensic Science SA, Adelaide, Australia over a 10-year period from 1994 to 2004 where significant decomposition had been documented. The age range of victims was 10 months to 92 years (mean 51.5 ± 18.1 years). There were 498 males (79%) and 131 females (21%) (M:F = 3.8:1). Deaths in 289 of the 629 cases (46%) were due to natural causes, with 179 suicides (28%), 83 accidents (13%), and 36 homicides (6%). The cause of death was ascertained in 89% of cases and the manner of death in 93% of cases. In 35 cases (6%) both the cause and manner could not be determined. Numbers of suicides were higher in younger age groups while deaths from organic illnesses predominated in later life. The number of cases in summer was significantly greater than in winter. Despite technical difficulties in handling decomposed bodies, and artifactual alteration of tissue structure and microscopic features, the autopsy was still a useful diagnostic exercise. While it is likely that more information may be gleaned from fresh bodies in perfectly preserved states, decomposed bodies may reveal significant anatomical and pathological features that enable both the cause and manner of death to be established.     

Measuring temporal expression,systematic response,and post-mortem stability to assess potential markers for estimating wound age: an example of Fosl1 in contused skeletal muscle

To assess whether Fosl1 is a suitable parameter for wound age estimation, a total of 126 male Sprague–Dawley rats were divided into control (n = 6 per group), contusion (including 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 h post-severe injury, and 4 or 8 h post-moderate or -mild injury subgroups), and post-mortem (including 6, 12, 18, and 24 h subgroups) groups (n = 6 per subgroup). A contusion was produced in the right limb of the rats using the drop-ball technique (under intraperitoneal chloral hydrate anaesthesia), and the animals were sacrificed using a lethal dose of pentobarbital. The expression of Fosl1 mRNA and protein was determined in the contused and contralateral uninjured muscle and post-mortem specimens of musculi quadriceps femoris using real-time PCR and western blotting, respectively. Compared with the control group, the expression of Fosl1 mRNA was increased in several contused and contralateral uninjured muscles, and decreased in post-mortem groups (P<0.05). The expression of Fosl1 protein was increased in the 4, 8, 32, 36, 40, 44, and 48 h contusion subgroups (P<0.05), but there was no significant change in the contralateral uninjured muscles and post-mortem specimens. The results indicated that Fosl1 protein was more specific and stable than Fosl1 mRNA, which suggests that the temporal expression, systematic response, and post-mortem stability should be comprehensively analysed when exploring other markers for use in wound age estimation in the future.