enzyme histochemistry


Introduced in the 1960s by Raekallio, time-dependant changes in the activity of various enzymes at wound edges were evaluated, predominantly in experimental animals. The diagnostic value of these extrapolations has been questioned, however, as positive findings do not occur as consistently in humans as they do in experimental animals (Betz 2003).



The detection of fibrin was thought to be proof of vitality, but can be deposited at the site of injury up to 6 hours after death. The morphological features of 'post mortem fibrin' are difficult to interpret.

Researchers have examined the relationship between cell-adhesion molecules (including  the selectins P-selectin and E-selectin, as well as ICAM-1) and wound vitality. P-selectin positivity is increased in vital wounds, with strong staining visible in endothelial cells after a few minutes, and remains visible for up to 7 hours. Positivity for E-selectin is seen after 1 hour (up to 17 days).

Fibronectin (involved in leucocyte migration) immunohistochemistry, and evaluation of proliferation markers (such as Ki-67) and apoptoses have also been evaluated in the recent past.

Articles reviewing immunohistochemical parameters for wound age estimation;


  • Betz (1995)
  • Takamiya M et al (2002)
  • Akasaka Y et al (2004)
  • Suarez-Penaranda et al (2002)
  • Kondo et al (2002a)
  • Kondo et al (2002b)
  • Fiegulth A et al (1997)
  • Tarran et al (2006)(burns)
  • Cecchi R (2010)

practical evaluation


Betz (2003) provides a useful summary of how to approach wound age estimation, given the vast amont of data available, much of which is based on experimental wound infliction, often on animals;

  • the detection of fibronectin and interstitial collagens (with network-like structures distant from the wound margins and bleeding zone) is significant;
  • the collagen response must be associated with fibroblast proliferation;
  • negative and positive controls must be provided;
  • multiple specimens from each wound need to be evaluated if a 'negative' finding is being relied upon.

Cecchi (2010) advises that the forensic pathologist should 'select the most appropriate markers' to be identified, according to the technology available in his/ her institute as well as the 'estimated timing' of the inflicted injury. For example, if it is thought that the wound is of the order of 2-3 hours, markers of endothelial activation might be appropriate (ICAM-1 and VCAM-1). Wound vitality markers of interest are those which apparently are 'never' or 'constitutionally scarsely' present (in human skin) such as Selectin-E and IL-1B, or are 'known to be absent' in post mortem wounds such as Fibrin D-dimer and Cathepsin A and D.

Future fields of research are likely to extend into the proteogenomics/ geno-proteomics of the inflammatory response, and Cecchi (2010) identifies NFkB activation as a key marker of interest.

histological changes


 source: wikimedia


polymorph neutrophils

Although the presence of neutrophils is assumed to represent a vital reaction, it should be noted that they can reach damaged tissue passively, and only their presence beyond the bleeding zone should be regarded as positive evidence of migration. Similarly, margination of neutrophils in vessels may be due to artefactual attachment, whilst permeation of vessel walls is assumed to be a vital process (Betz 2003).


  • earliest vital neutrophil reaction - 15-30 mins post wound infliction;
  • routinely described as appearing after 15 hours; and
  • have been described several months after wound infliction.

Therefore one can only say that a wound without neutrophils indicates a post-infliction period of less than 15 hours.



Migration of macrophages to the area of damage occurs at the same time as neutrophils, but their reduced motility delays their appearance.

  • earliest detection - 3 hours post infliction;
  • usually found 3 days+ after wounding;
  • a macrophage excess over neutrophils occurs at the earliest 20 hours post injury and routinely after 11 days.

Lipophages (macrophages incorporating fat) and erythophages (macrophages incorporating red blood cells) can be seen 3 days post wounding.

Haemosiderin (formed during the degradation of haemoglobin) can be detected by the Perl's stain (Prussian Blue) after approximately 3 days post wounding (Betz 2003).

(See also McCausland and Dougherty (1978) for a study of the histological ageing of bruises in lambs and calves)


granulation tissue and re-epithelialisation

This can be detected approximately 3 days+ after wounding. Complete re-epithelialisation in surgical wounds takes at least 5 days, and is routinely found after 21 days.



estimation of wound vitality and 'age' of wound


Traumatic insults to the skin result in an acute inflammatory response, and researchers in forensic pathology have attempted to define the characteristics of a wound inflicted during life (a 'vital' injury) as opposed to one inflicted after death.

Hernandez-Cueto et al (2000) re-produce Legrand du Salle's 'schema' for the diagnosis of vital and post mortem wounds;


  Vital wounds Post mortem wounds
Wound edges
  • initially - swollen, hard, separated due to tissue retraction, blood infiltration
  • later - lymph exudation and suppuration
  • not swollen, soft, closed together and not retracted


  • lack of lymph exudation or suppuration
Surrounding tissues


  • abundant haemorrhage infiltrating tissues
  • lack of bleeding/ infiltration
Inside the wound
  • coagulated blood
  • lack of coagulated blood


(Source: Adapted from Hernandez-Cueto et al (2000))



histological parameters in age estimation of human skin wounds (Betz 2003)


Parameter Earliest appearance Routine appearance Latest appearance
neutrophils 15-30 min >15 h months
macrophages 2-3 h >3 days months
macrophages>neutrophils 20 h >11 days months
migrating keratinocytes 2 days >9 days -
lipophages 3 days - months
erythrophages 3 days - months
siderophages/ haemosiderin 3 days - months
granulation tissue 3 days - months
complete re-epithelialisation 5 days >21 days -
hematoidin 8 days - months
lymphocyte-infiltrates 8 days - months

schema for the histological estimation of the age of open skin wounds and abrasions (Raekellio 1980)


Survival period Findings
<4 hours
  No distinct signs of inflammation
  histological distinction between ante and post mortem not possible
4-12 hours
  4 hours - some perivascular neutrophils;
  8-12 hours - neutrophils, macrophages, activated fibroblasts form 'peripheral wound zone' (neutrophils >macrophages (5:1))
12- 48 hours
  16-24 hours - neutrophils: macrophages 0.4-1. After 16 hours - fibrin red with Martius Scarlet Blue (yellow before 16 hours)
  At 24 hours - neutrophil numbers and fibrin peak (for 2-3 days); cut edge of epidermis shows cytoplasmic processes
  24- 48 hours - epidermis migrates from incised edge towards centre of wound
  At 32 hours + necrosis apparent in 'central wound zone'
  At 48 hours macrophages reach maximum concentration in peripheral zone
2- 4 days
  At 2- 4 days - fibroblasts migrate from the nearby connective tissue to wound periphery
  At 3 days - epithelialisation of small wounds and abrasions complete; thereafter regenerated epidermis becomes highly stratified and thicker than normal surrounding epidermis
  At 3- 4 days - capillary buds appear
4- 8 days
  At 4 days - first new collagen fibres seen
  At 4- 5 days profuse in-growth of new capillaries; capillaries continue to proliferate until 8th day
  At 6 days - lymphocytes reach maximum concentration in wound periphery
8- 12 days
  Decrease in number of inflammatory cells, fibroblasts and capillaries; increase in the number and size of collagen fibres
>12 days
  Definite stage of regression of cellular activity in both epidermis and dermis. Vascularity of dermis diminishes. Collagen fibres restored. Epithelium shows stainable basement membrane.
  At 14 days - fibroplasia reaches peak. Thereafter there is gradual shrinkage and maturation of connective tissue in the wound

immunohistochemical parameters in age estimation of human skin wounds (Betz 2003)


Antigen   Earliest appearance Routine appearance Latest appearance
P-selectin   minutes - 7 h
fibronectin   10-20 min >4 h months
E-selectin   1 h - 17 days
ICAM-1   1.5 h - 3.5 days
fibroblast proliferation   1.5 days >6 days -
fibroblast apoptosis   1-2 days - -
tenascin   2 days >5 days months
collagen III   2-3 days >6 days months
collagen V   3 days >6 days months
collagen VI   3 days >6 days months
collagen I   5 days >6 days months
  laminin 1.5 days - months
  HSPG 1.5 days - months
  collagen IV 4 days - months
  α actin 5 days - months
basement membrane        
  bm-fragments 4 days >13 days >21 days
  bm complete 8 days >21 days -
macrophage subtypes        
  RM 3/1 7 days - -
  25 F 9 11 days - -
  G 16/1 12 days - -
complete staining of the epidermis for keratin 5   13 days >23 days  




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