Skin wounds vitality markers in forensic pathology: An updated review

Abstract

Wound age evaluation is one of the most challenging issues in forensic pathology. In the first minutes or hours, standard histological examination may not determine whether the wound was inflicted in the pre- or post-mortem period. While red blood cell infiltration is classically considered as a sign of vital reaction, several studies have shown that extravasation of blood cells may also occur after death and cannot be used as a reliable marker in the diagnosis of wound vitality. Numerous studies about wound vitality are available in the literature. They have evaluated markers involved in coagulation or inflammation, using various methods such as enzymology, molecular biology or immunohistochemistry. In this update, we first introduce some methodological principles. Then, we review the main studies available in the literature. Immunohistochemistry seems to be the most valuable method, given its easy application and the possibility to analyse the localization of the molecules of interest. Some markers are promising, such as CD15, TNFα, IL-6, IL-1β, TGFα or TGFβ1. Prior to their application in daily practice, these early results need to be confirmed with other studies, conducted by independent teams and integrating multiple controls. Most notably, the antibodies have to be tested in numerous post-mortem wounds. Indeed, a critical risk of overexpression in post-mortem wounds is present. Some promising markers have been later invalidated because of post-mortem false positivity. Finally, optimal sensitivity and specificity values could probably be reached by combining several markers, validated by large groups of pre- and post-mortem wounds.

Keywords

Wound vitality marker immunohistochemistry forensic pathology

Introduction

In medico-legal investigations, wound age estimation remains a major concern in many situations. In cases of violent death, skin wounds can be sampled during autopsy to determine the time interval between trauma and death. Most notably, the forensic pathologist has to distinguish ante-mortem from post-mortem wounds, and try to evaluate the survival time of the victim.

In the first minutes or hours, standard histological examination may not be able to determine whether the wound was inflicted in the pre or post-mortem period. While red blood cell extravasation is classically considered as a sign of vital reaction, several studies have shown that extravasation of blood cells can also occur after death and does not represent a reliable marker in wound vitality diagnosis.^1–3 Thus, the presence of infiltration of polymorphonuclear neutrophils (PMN) is to date the only reliable histological criterion to differentiate recent pre-mortem from post-mortem wounds.

Scientific studies in this field deal with research on relevant markers of vital origin, using ancillary techniques such as enzymology, immunohistochemistry or molecular biology. To date, no marker has proven to be efficient and reliable. Indeed, the results are contradictory, due to a lack of reproducibility or inadequate diagnostic performance, while some studies are still preliminary and lack sufficient cases and controls.^4–6 Some new markers seem to be promising. However, additional research with adequate large case series, controls and independent investigators are needed.

In this review, we first introduce some methodological principles. Then, we investigate the most promising markers and their potential usefulness in forensic applications.

Pathophysiology of healing

The wound healing process includes sequential phases and concerns skin injury as well as other organ damage.^7–9 Blood clot formation initiates this process. This is followed by inflammation, in order to recruit leukocytes and prevent infection. During this inflammatory process, destruction of damaged tissue occurs via necrosis or apoptosis. The recruited scavenger cells remove debris and achieve tissue debridement. Then, the regeneration phase starts with granulation tissue formation at the wound site. Finally, a new epithelium replaces granulation tissue, and this newly formed tissue matures to cover the defect and form a new scar.^9,10

These distinct phases more or less overlap. Numerous factors can influence the healing process,^4,11 such as age,^12,13 pre-existing disease¹¹ and previous medication or drug consumption.^14,15 Moreover, wound localization¹² and the circumstances surrounding the injury must be scrutinized, together with the environmental conditions the corpse was exposed to and post-mortem artefacts.¹¹ Finally, tissue sampling and laboratory procedures can influence the analysis.

Various investigators have described age determination of injuries using standard histological criteria through this wound healing chronology. Moreover, the release of several mediators (coagulation factors, cytokines, growth factors, etc.) by damaged tissue may add some promising insight to identify relevant indicators.

Conventional histology

Haemorrhagic infiltration is considered as a vital sign, but some studies have revealed that red blood cell extravasation can also occur after circulatory arrest^1–3 (Figure 1(a)). The presence of inflammatory cells is the only solid standard histological finding of ante-mortem origin.^11,16 Granulocytes are the first cells to migrate to the wound site. First observations of PMN vary between 10 min and 12 h, and in most cases are within 1–2 h.^6,7 PMN reaction starts with cell marginalization on the endothelium and diapedesis across the vascular wall. Thus, evidence of infiltration of granulocytes must be initially evaluated on small vessel areas and perivascular tissue (Figure 1(b)).

Figure 1.

(a) Standard histological examination showing haemorrhagic infiltration (arrow) in the margin of a post-mortem wound. (b) Perivascular polymorphonuclear neutrophils (arrowhead) in a recent pre-mortem stab wound (haematoxylin, eosin and saffron, ×400).

Other inflammatory cells can provide additional information for post-traumatic interval estimation.^6,7,11,17 Indeed, monocytes migrate into the wound region after extravasation and thereafter change into phagocytic macrophages. A 4–8 h delay before macrophages appear can be observed. Siderophages (macrophages with deposition of intracellular siderin) occur after 1–2 days and haematoidin deposits are present after approximately 15 days. Infiltrating lymphocytes are clearly visible 2–3 days after injury. Nonetheless, pathologists must keep in mind that basal inflammatory cells can be observed in non-injured tissue.

Standard histological findings must be dealt with carefully due to large inter or intra-individual variations. In recent wounds, it is possible for no PMN to be observable for several minutes or hours after injury. Consequently, affirmation in pre-mortem or post-mortem injury requires further ancillary techniques.

Ancillary studies: Methodological principles

A large number of substances are secreted during the healing process. In particular, markers involved in the first steps of wound healing (haemostasis, initiation of inflammation) must be taken into consideration as effective in the diagnosis of wound vitality. Early detection of these relevant markers reflects the release of newly formed or preformed molecules.

The studied parameters have to be absent in physiological conditions or significantly increased at wound site. Quantitative or semiquantitative evaluation is performed in this latter case, and a reference threshold should be determined versus internal control.⁴ The margin of error must be statistically evaluated by determination of a confidence interval, sensitivity, specificity, and positive and negative predictive values. A large number of samples is needed to reduce the confidence interval. The proof of value of a marker is established with sensitivity and specificity: it is essential to obtain the smallest number of false-negative and false-positive cases. In order to use a marker in a legal context, sensitivity and/or specificity should be virtually 100%. In addition, appropriate use of numerous negative and positive controls is needed to validate the diagnostic value analysis and provide solid information.¹⁸ Furthermore, the smallest time interval for detection of a marker must be precisely established in order to document the minimum wound age.⁴ When using a morphological method, analysis should also be performed independently by two investigators in order to evaluate inter-observer reproducibility.

Numerous methods can be used to detect these indicators: study of mRNA (RT-PCR, in situ hybridization), protein (ELISA technique, Western blot) or morphologically oriented techniques such as immunofluorescence and immunohistochemistry.^6,18,19 In forensic pathology, immunohistochemistry is the method of choice due to its reliability and easy application in formalin-fixed paraffin-embedded tissue.²⁰ Contrary to most techniques, this morphological method enables location of the substance of interest within the tissue or cell substructures.

Several scientific studies have been published about vitality markers. The choice of samples is critical. Studies are designed according to three research models.⁴ First, autopsy samples appear to be the most accurate and realistic model, especially if survival time is known. This information is nonetheless frequently absent or insufficiently documented. The circumstances of wound infliction also represent an influencing factor; moreover, strict sample selection is required. For instance, comparing a stab wound with a blunt force injury can be challenging. Second, research models with surgical samples can involve controlled and standardized conditions. However, results are not strictly similar to reality, due to anaesthesia, drug administration or stress conditions.¹⁸ Animal experiments have the advantage of fully controlled conditions,^21,22 but the results must be confirmed with human tissue data before application in forensic pathology.

Immunohistochemical marker evaluation on post-mortem wounds is crucial in order to exclude supravital artefactual labelling.^23,24 If the wound is sampled perpendicularly to its margin, marker expression can be compared with the opposite margin as an internal post-mortem control.¹⁸ During autopsy, another possibility is to sample control tissue on the median thoraco-abdominal incision margin. However, post-mortem control wounds performed during autopsy, often several days after death, are less relevant than wounds inflicted a few minutes after death. Indeed, a long interval between injury and death could reduce the risks of artefactual post-mortem blood extravasation and hypothetical supravital chemotaxis.²⁵ While multiple studies have proposed animal models for the evaluation of injury inflicted in the early post-mortem interval,²⁶ we devised an original ex-vivo human model of recent post-mortem wounds, consisting of injuries inflicted to surgical specimens, 5 min after devascularization.²⁷

Factors involved in haemostasis balance

The first phase of haemostasis begins with vascular constriction immediately after wound infliction. Activated endothelium produces vasoconstricting factors such as amines in order to limit haemorrhage and red blood cell extravasation. Primary haemostasis leads to platelet plug formation and the release of numerous substances such as histamine, serotonin, thromboxane, prostaglandin, platelet activating factor (PAF), platelet-derived growth factor (PDGF), and growth factors or cytokines (TGF-β, IL-1, TNF). Most of these factors are also involved in the initiation of the inflammatory process.²⁸

Secondary haemostasis involves blood clot factors and cofactors in the coagulation cascade which convert fibrinogen under the effect of thrombin. The end result of these sequential reactions is a stable clot with cross-linked fibrin strands.

Due to their early release or production, substances present in haemostasis could be valuable markers for the vitality diagnosis. However, in the field of forensic pathology, few authors have studied factors dealing with haemostatic balance. Two key markers are documented in the literature: fibrin and D-dimer. Fibrin is present at the wound site a few minutes after wounding. Although fibrin appeared to be a suitable marker of vital origin in several studies,^18,29,30 some research based on immunohistochemistry has documented fibrin positivity in post-mortem injuries.^18,29,31 D-dimer, a degradation product of the fibrin network, could be a wound vitality marker, as described in an ELISA test study using frozen tissue.³² Evidence of a statistically significant difference was provided only with incised skin wounds, and not in abrasions and contusions. However, the sensitivity gain compared with histology is not described and the time interval between wound and death is not specified. Further studies about this marker are needed.

In addition to these substances, Van de Goot⁷ reported overexpression of factor VIII (FVIII) in endothelial cells in vital injuries with the immunohistochemistry technique. In this study, the strong endothelial overexpression observed on the figure and the absence of other references to FVIII in the literature lead us to believe that this marker was probably FVIII-related antigen (FVIIIra, also known as von Willebrand factor). Indeed, a study on cerebral lesions described endothelial cells stained with FVIIIra antibody within the damaged tissue, identified as early as 3 h after trauma.³³ In normal tissue, FVIIIra showed a weak or non-staining reaction. Recently, we demonstrated a strong interstitial staining for FVIIIra, but no endothelial upregulation at the skin wound margin of vital stab injury.²⁷ In our study, we noted a 100% sensitivity, but we found a FVIIIra overexpression in numerous post-mortem controls, leading to a 47% specificity. Thus, this marker does not seem useful for establishing an intravital injury diagnosis.

Adhesion molecules

Cell adhesion molecules are proteins involved in the wound healing process. Expression of these substances by endothelial cells allows interaction with leukocytes, such as rolling, adhesion and diapedesis; they are critical in the initial inflammation phase. In the regeneration phase, adhesion proteins are more important for cell migration in cells such as such as fibroblasts or keratinocytes.

Among these molecules, the intercellular adhesion molecule 1 (ICAM-1) and the vascular cell adhesion molecule 1 (VCAM-1) were reported to be expressed by endothelial cells at the injury site. However, ICAM-1 and VCAM-1 were not detected immunohistochemically until 1 h 30 min and 3 h, respectively.^5,26 Expression of selectins seemed to be promising when considering the previous findings.^5,18,26,34 The time interval detection for E-selectin varied between skin wound age of 1 h to 17 days; therefore, this marker does not appear more efficient than standard histology in the diagnosis of skin wound vitality. Interestingly, P-selectin was described to be stained as early as 3 min after injury. However, Ortiz-Rey et al. described staining for P-selectin in post-mortem skin samples and concluded that P-selectin is not a specific wound vitality marker.³⁵

CD15 antigen is expressed in leukocytes, most notably PMN and activated monocytes, and is involved in cellular adhesive events. Indeed, CD15 contributes to leukocyte adhesion through interaction with endothelial surface selectins.³⁶ Hausmann et al.³⁷ analysed CD15 expression in human brain contusions by immunohistochemistry. PMN staining began as early as 10 min after brain damage. Turillazzi et al.³⁸ confirmed CD15 granulocyte expression in vital hanging marks but not in post-mortem controls. In stab wound injuries, we showed a significant CD15 overexpression in a vital wound, with a 9 min minimum time interval.²⁷ The sensitivity was 47% (Figure 2). Interestingly, CD15 was not significantly stained in control or post-mortem samples, leading to a 100% specificity. Inter-observer variability was high, reaching 0.90.

Figure 2.

CD15. Multiple perivascular polymorphonuclear neutrophils staining for CD15 (arrow) in a vital surgical incision (immunohistochemistry, ×400).

Several studies have described fibronectin as a wound vitality marker.^39–42 Fibronectin could be detected immunohistochemically with a survival time of a few minutes.^39–42 However, Grellner reported that fibronectin staining was positive in a post-mortem incised wound in a porcine skin model.⁴³

Chemokines, cytokines and pro-inflammatory substances

Chemokines and cytokines are chemical mediators and have an active role in extracellular communication pathways. These regulatory proteins are involved in the inflammatory phase and wound repair process by recruiting leukocytes. In this way, PMN infiltration is triggered by the chemoattractive properties of these substances, such as leukotriene B4 (LTB4), PAF and interleukin 8 (IL-8).⁴⁴ Furthermore, as these markers are stored or newly synthesized with short half-lives, their expression should occur in the early post-traumatic interval.

To date, the available data indicate a minimum time for detection of these substances of more than 4 h after injury, which is probably not suitable for the diagnosis of wound vitality.⁵ Protein levels could be too low in the first minutes following the wound for effective detection with current techniques.

LTB4 is produced as a result of leukocyte membrane phospholipid metabolism. He and Zhu⁴⁵ reported an LTB4 upregulation in ante-mortem skin wounds using high-performance liquid chromatography (HPLC). In order to apply the HPLC technique, the duration of formalin fixation of samples should not exceed 10 days. However, the minimum time interval for positivity is not detailed in this study. Larger series of specimens are needed to confirm these results and to evaluate whether LTB4 is suitable for wound vitality estimation. The authors did not provide evidence of overexpression of other leukotrienes (LTC4, LTD4) or prostaglandins. Prostaglandins, such as prostaglandin F2a (PGF2a), seem irrelevant to study skin wound vitality. Indeed, Hernández-Cueto et al.⁴⁶ concluded their investigation by mentioning the technical difficulties and disadvantages of PGF2a analysis, such as high cost, numerous influencing factors and sample-handling protocol.

PAF is expressed by activated endothelial cells and is involved in the early inflammatory response and haemostasis. To our knowledge, in forensic pathology, no positive results concerning this potentially interesting substance have yet been reported.¹⁸

Tumour necrosis factor alpha (TNFα) could be a helpful tool for wound vitality estimation. Bacci et al.⁴⁷ reported a significant increase of TNFα-positive mast cells 15 min after injury, using immunofluorescence. Furthermore, Grellner et al.,^48,49 using immunohistochemistry, depicted staining of epidermis cells after 15 min survival, reaching a maximum intensity after 60–90 min. Other investigations are required to confirm these results.

Interleukin 1 beta (IL-1β) is a pro-inflammatory cytokine that has been highlighted early after trauma. Bai et al.⁵⁰ reported an IL-1β mRNA overexpression occurring approximately 30 min after trauma, and a peak level at 2 h, using a real-time fluorescent quantitative polymerase chain reaction (PCR). Grellner et al.,⁴⁹ using immunohistochemistry, pointed out the first IL-1β upregulation after 15 min and a higher expression between 30–60 min. Cell signalling appeared first in the injured epidermis germinative layers and then in granular layers. Normal skin epidermis demonstrated negative or less than 25% labelling of germinative layer cells. Finally, Grellner et al. supplemented their work with evaluation of interleukin 6 (IL-6), which was overexpressed in the epidermis after 20 min, with a peak between 60 and 90 min. TNFα, IL-1β and IL-6 were potent markers for wound age estimation. Larger series including analysis of negative controls, most notably post-mortem skin samples, are needed to validate these markers and to exclude supravital staining.

Takamiya et al.⁵¹ studied eight cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFNγ and TNFα) with a multiplex bead-based immunoassay. Investigators separated cytokines into three groups according to their time-course expression: early phase (IL-10, GM-CSF, IFNγ, TNFα), middle phase (IL-6), middle and late phases (IL-2, IL-4, IL-8). Data obtained are not directly applicable to practical forensic pathology, but these markers could be potential immunohistochemical targets for future investigation. Most notably, an experimental study in mice⁵² showed enhanced IL-10 immunoreactivity, with epidermis cells labelling 1–3 h after incision. Ohshima et al. observed IL-10 mRNA increasing after 15 min, with reverse transcriptase-PCR.⁵³

Vasoactive peptides and enzymes

Histamine is a vasoactive amine released by basophils and mast cells. In acute inflammatory reaction, histamine induces vasodilatation, vascular permeability increase and leukocyte extravasation. Various studies showed an excessive technique-dependent variability. With the HPLC technique, Raekallio et al.⁵⁴ reported an overexpression after 15–30 min, while the immunohistochemical histamine level increased up to 100% after 60 min.⁵⁵ An experimental study with a murine model, using the microfluorometric method, indicated that the skin histamine level was upregulated after 30 min. No statistical relationship was found between the mast cell number and histamine level.⁵⁶ To date, histamine is not a reliable marker in forensic pathology.

Tryptase is a serine protease predominantly expressed in mast cells and is an important mediator in anaphylactic and anaphylactoid reactions.⁵⁷ In addition to these acute processes, tryptase is a known mitogen for dermal fibroblasts. Bonelli et al.^58,59 used anti-tryptase and chymase antibodies to evaluate mast cell density in skin wounds by immunofluorescence. The dermal mast cell number increased progressively within a few hours from trauma (peak at 1–3 h). However, influencing factors such as post-mortem protein release from mast cells need to be excluded. In skin marks resulting from hanging, Turillazzi et al.,³⁸ using immunohistochemistry, noticed an overexpression of tryptase in interstitial tissue. These findings could reflect mast cell degranulation in vital ligature marks. Similarly, in recent wound margins, we observed an increase of mast cell degranulation rate (Figure 3), without significant difference in the number of tryptase-positive cells,²⁷ reaching 60% sensitivity and 100% specificity. However, the degranulation rate was relatively difficult to evaluate, leading in our study to a poor inter-observer agreement coefficient (0.42).²⁷ Finally, using the naphthol AS-D chloroacetate esterase (NAS-DClAE) technique, Oehmichen et al.⁶⁰ also stressed the significance of mast cell degranulation at intravital wound edges.

Figure 3.

Tryptase. Mast cell degranulation within surgical wound margins, identified by punctiform signals (arrow) adjacent to positive mast cells (immunohistochemistry, ×400).

Serotonin is a monoamine and a neurotransmitter, widely distributed in the central nervous system. In the periphery this neuromodulator, stored notably in platelets and leukocytes, can affect nociceptors and trigger a pain sensation. With the HPLC technique, an expression gain after 10–30 min was reported. Immunohistochemical serotonin staining was observed as early as 5 min, but was transient and extinct after 15 min.^18,61–65 In forensic pathology, serotonin use would thereby be restricted.

Cathepsins are lysosomal enzymes localized in inflammatory cells. Cathepsin A and D have been studied as potential vitality markers.^66–68 Experimental studies in pigs and humans, using spectrophotometry, noted an upregulation of enzymatic activity of cathepsins a few minutes after a skin wound was received. Investigators emphasized the expected benefits of cathepsin D. In practice, the use of this marker would need further investigation.

Growth factors and wound healing-related molecules

During the skin wound healing process, growth factors are essential for regulating and stimulating tissue repair. These molecules are present in most healing steps: angiogenesis with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), proliferation of keratinocytes with epidermal growth factor (EGFs) and transforming growth factor α (TGFα), proliferation of fibroblasts and extracellular matrix production with TGFβ1 and fibroblast growth factor (FGFs).^5,6,18,69

TGFα and TGFβ1 are the most promising markers in this group of molecules. Indeed, Grellner et al.⁷⁰ investigated their relevance for skin wound age estimation. On the one hand, TGFα-positive cells were observed between 10 and 20 min, with a stronger reaction between 30 and 60 min after wound infliction. Staining involved spinous layer keratinocytes (median epidermis layers). Normal skin did not react to TGFα antibody, or reacted only slightly. Skin control investigation from the same individual needs to be performed to eliminate variations in basal expression. No information was provided on post-mortem control staining; therefore, these interesting results must be confirmed.

On the other hand, TGFβ1 was also analysed by Grellner et al. and they observed an overexpression of TGFβ1 after several minutes at the earliest, with a peak between 30 and 60 min. Pattern staining concerned basal and spinous epidermis layers, primarily in traumatized skin areas. Reactivity was stressed on haemorrhagic regions and in the upper dermis. However, the authors were not totally convinced of the reliability of this marker in post-mortem injuries: the reaction might also be positive in post-mortem wounds with consecutive bleeding. Indeed, other investigators had previously reported post-mortem TGFβ1 positivity.⁷¹

In the course of wound healing, many other substances are taken into account such as extracellular matrix components (collagen types III, IV, V and VI, laminin, etc.) or specific cell type markers such as smooth muscle actin for myoid cells (myofibroblasts, myocytes, etc.).^5,18 These substances do not seem to be relevant for wound vitality assessment, because of their late expression, after several days.

Discussion

To date, different techniques may be applied to forensic specimens to estimate wound age. Forensic pathologists probably consider immunohistochemistry as the most suitable method due to its easy access and use with formalin-fixed paraffin-embedded tissue. Moreover, this morphological method allows marker localization to be pinpointed accurately within the tissue or cell substructures.

Various studies on the expression of cytokines such as TNFα, IL-1β and IL-6 or growth factors such as TGFα have shown promising results, with positive staining within the first minutes after wound infliction (Table 1). Other independent investigations are required to confirm these results before their use in daily practice. Furthermore, to exclude supravital reactions it is necessary to evaluate markers on post-mortem wounds. Indeed, as markers should present an optimal specificity in order to be used in legal procedures, the risk of false positivity is critical. Some substances, such as fibronectin or P-selectin, are unreliable due to false-positive findings in post-mortem cases. One of the main standard criteria in the evaluation of an immunohistochemical marker is its inter-observer reproducibility. However, no data about reproducibility was found in most studies.

Table 1.

Main immunohistochemical vitally markers studied in forensic pathology (paraffin-embedded specimens). False positivity is defined by overexpression in post-mortem wounds and false negativity by lack of expression in wounded skin samples.

Marker	Location	Delay	Samples (n)	Controls (n)	False-positive	False-negative	Reference
FVIIIra	Interstitial	ND	Autopsy (12) and surgery (58)	Post-mortem lesions (8) and surgical control samples (24)	Yes (17/32)	No	Gauchotte, 2013
E-selectin	Vascular	1h	Autopsy and surgery (197)	Post-mortem lesions (31)	No	Yes (96/197)	Dressler, 1999
	Vascular	1h	Autopsy (194), surgery (100), murine model (140)	Post-mortem lesions (31)	ND	ND	Dressler, 2000
P-selectin	Vascular	3 mn	Autopsy and surgery (197)	Post-mortem lesions (31)	Yes (4/31)	No	Dressler, 1999
	Vascular	3 mn	Autopsy (194), surgery (100),murine model (140)	Post-mortem lesions (31)	ND	ND	Dressler, 2000
	Vascular	ND	Surgery (24)	Post-mortem lesions (14)	Yes (ND)	ND	Ortiz-Rey, 2008
CD15	Leukocytes	9 mn	Autopsy (12) and surgery (58)	Post-mortem lesions (8) and surgical control samples (24)	No	Yes (37/70)	Gauchotte, 2013
Fibronectin	Interstitial	Few mn (min), 30 mn (max)	ND (53)	Post-mortem lesions (6)	No	Yes (7/53)	Betz, 1992
	Interstitial	Few mn	Autopsy (13)	Post-mortem lesions (13)	Yes (ND)	No	Betz, 1993
	Interstitial	ND (post-mortem)	Porcine model	Post-mortem lesions (36)	Yes (18/36)	ND	Grellner, 1999
TNFα	Mast cells	15 mn	Autopsy (40)	Post-mortem lesions (10) and surgical biopsies (10)	No	Yes (ND)	Bacci, 2006
	Keratinocytes	15 mn (min), 60-90 mn (max)	Autopsy and surgery (105)	Internal control * (105)	ND	Yes (ND)	Grellner, 2002
IL-1β	Keratinocytes	15 mn (min), 30–60 mn (max)	Autopsy and surgery (105)	Internal control * (105)	ND	Yes (ND)	Grellner, 2002
IL-6	Keratinocytes	20 mn (min), 60–90 mn (max)	Autopsy and surgery (105)	Internal control * (105)	ND	Yes (ND)	Grellner, 2002
Tryptase	Mast cells	1 mn	Autopsy (12) and surgery (58)	Post-mortem lesions (8) and surgical control samples (24)	No	Yes (28/70)	Gauchotte, 2013
TGFα	Keratinocytes	10 mn (min), 30–60 mn (max)	Autopsy and surgery (74)	Internal control * (74)	ND	Yes (33/74)	Grellner, 2005
TGFβ1	Keratinocytes	Few mn (min), 30–60 mn (max)	Autopsy and surgery (51)	Internal control * (51)	ND	Yes (5/51)	Grellner, 2005

h: hour; mn: minute; ND: not documented; min: minimum time interval; max: maximum time interval; *: uninjuried site in same specimen.

Another important point is to evaluate the reliability of these markers in altered specimens. Indeed, putrefaction is frequent in the medico-legal context, and in this situation standard histology often fails to detect morphological findings such as infiltrating inflammatory cells. It is particularly difficult in this field to obtain an experimental protocol that can be applicable to forensic diagnostic pathology.¹⁸ Animal corpses could be used, with results comparable with natural decomposition, but these findings would need confirmation in human specimens. Controlled ex-vivo putrefaction can be used with human tissues, although this is not totality comparable with human whole-corpse putrefaction, notably because microbial flora composition and putrefaction gases cannot be strictly transposed to ex-vivo protocols. We previously tested an ex-vivo desiccation and putrefaction protocol, showing the unreliability of immunohistochemistry in altered specimens.²⁷ We subjected human skin samples at 1, 2 and 3 weeks to desiccation in an open air bottle, and to artificial putrefaction in human blood obtained from colectomy specimens. However, staining for tryptase and CD15 could not be analysed with accuracy in these altered samples.

In conclusion, although multiple studies about vitality assessment are available in the literature, few markers seem to be sensitive and/or specific enough, as well as easily identifiable, for daily use in forensic pathology. To date, no marker has been definitely validated, because of positive and negative control numbers that are too small, or due to the absence of evaluation of inter-observer reproducibility. Finally, combining several markers would probably contribute to sufficient sensitivity and specificity for using these analyses as evidence in court.

Footnotes

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

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