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).

immunohistochemistry

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.