There are many publications on this subject, as it is a favourite topic for research. As with time of death, it can be an important matter in forensic medical investigations to determine whether a wound found at autopsy was inflicted before or after death and, if inflicted ante-mortem, how long before death it was sustained.
Unfortunately, as with so many problems, biological variability introduces a wide margin of uncertainty, so that a range of probabilities can be offered, but never a definite time interval. Much of the experimental work has been performed on animals and the results are not transferable to man – a common defect in all animal work. The smaller FIGURE4.54 Defence injuries: bruising on the back of the hand in
an attempt to fend off a blunt instrument in this case a metal poker that was used to inflict fatal head injuries. Such injuries confirm that the victim was conscious and active during the attack, and that it was not made covertly while the victim was unaware.
FIGURE4.53 Multiple homicidal stab wounds by closed scissors.
the animal, the more rapid the tissue scavenging and repara- tive changes: more primitive animals have far greater powers of regeneration than man, both in wounds and in regrowing organs and even limbs.
The changes will also vary according to the size of a wound, the type of wound (bruise, abrasion, incision or laceration), the tissue (epidermal or mesodermal), whether there is infection and the age and health of the victim.
The subject is complex and often contradictory and the following schedule of events is offered only as a guideline.
The original papers should be consulted if any particular criterion is to be followed; histological appearances, including fluorescence studies may be backed up by histo- chemical and biochemical assays. The sequence of changes in bruises is far less distinct than in abrasions or lacerations and histology is much less helpful. The most useful criterion
in a bruise is probably Perl’s reaction for haemosiderin, which becomes positive about 3 days after infliction – though some workers claim an earlier appearance, even down to 12 hours. Haemolysis of red blood cells is irregular and patchy, many intact cells being seen in some bruises after many days, so their rupture and ghosting cannot be used as an index of age.
Chronological histological changes after wound infliction
30MINUTES–4HOURS
Margination of polymorph leucocytes in dilated small ves- sels may occur, a feature which is often completely absent.
It is also not a reliable sign of ante-mortem infliction, as
Dating of wounds by histology and histochemistry
FIGURE4.55 Defence wound on the back of the hand from trying to ward off the knife.
FIGURE4.56 Typical defence injuries in a victim of a knife attack. In grasping the blade to deflect it, there have been cuts across the palmar surfaces of the finger joints and a slash between the thumb and forefinger, together with a cut at the base of the thumb.
leucocytes may congregate for many hours after death, especially in skin and around aspirated material in the lung.
Some extravascular emigration may begin at the end of this period, but a significant number of polymorphs usually delay for many more hours. Polymorphs tend to appear earlier in the subcutaneous fat than in the upper dermal layers. Basophilic ‘mast cells’ lose their granules. Fibrin appears in the wound within a few minutes, but this also occurs in post-mortem injuries.
4–12HOURS
Leucocyte infiltration is likely to be more definite, still mostly polymorphs, but some mononuclear cells as well.
A scanty mixed population of lymphocytes and poorly differentiated ‘monocytes’ appears, usually after 12 hours.
Tissue oedema and swelling of vascular endothelium occurs.
In small wounds involving the skin, the start of epithelial regeneration may be seen at the sides at the level of the basal layer of epidermis.
12–24HOURS
Leucocytes tend to demarcate the area of the wound by form- ing a marginal palisade. The polymorph response declines and the macrophage and mononuclear cell population increases from now on. Removal of necrosed tissue begins, with macrophages evident and a basophilic tinge to the ground substance. Mitoses are visible in fibroblasts from about 15 hours. The epidermis begins to spread across the surface of the scab and down the sides of a cut into the wound.
24–72HOURS
Leucocyte infiltration reaches a peak at about 48 hours;
repair begins concurrently with many fibroblasts appear- ing, but rarely before 72 hours. New capillaries then begin to bud from vessels, the vascular, infiltrated stroma becoming
‘granulation tissue’.
3–6DAYS
Repair is proceeding apace: collagen begins to form and giant cells may be visible around necrotic debris and foreign matter. The epidermis is actively growing if the wound involves the surface: in animals, it grows laterally at about 200 m/day. Haemosiderin becomes stainable from about the third day onwards, if there is a bruise or any bleeding into the wound, but is often not obtainable by Perl’s reac- tion until the fifth day even though some claim it can appear on the first or second day.
10–15DAYS
Cellular reaction subsides in small wounds. Vascularity decreases and the cell population drops, especially leucocytes.
Fibroblasts are most active, with collagen being laid down.
The epidermis becomes thin and flat, but no papillae (‘rete pegs’) are reformed for many weeks. Elastic fibres are scarce for a long time and less than in adjacent undamaged tissue.
TWO WEEKS TO SEVERAL MONTHS,DEPENDING ON SIZE AND OTHER FACTORS
Consolidation of the healing tissue continues. The inflam- matory response has vanished, unless the wound has become infected. Collagen and elastin increase and a vascular scar is formed, which gradually becomes more dense and avascular. The epithelium remodels and dermal papillae reappear unless the wound is wide and irregular. Skin adnexae do not reappear in the scar unless islands of viable skin survive within the wound area.
Histochemical changes in injured tissues
Histochemical and immunohistochemical methods have been studied widely in recent years, again mainly on animals, the transfer to the human situation being suspect in some cases. A wide variety of markers have been claimed as reliable for both the differentiation between ante-mortem and post- mortem injuries and for dating of ante-mortem wounds.
Electrolytes, such as sodium, zinc, magnesium and calcium, serotonin, esterases, glycophorin A and especially histamine have been used for this purpose.
FIGURE4.57 Defence wounds of hand in attempts to ward off an assault with a meat cleaver.
Survival period after wounding
Mainly as a result of the pioneering work of Jyrki Raekallio in Finland, the histochemical sequence of events in wounds has been actively pursued in recent years. Some techniques need frozen sections, others can be carried out on fixed tissue. Estimations of histamine or serotonin are usually ‘tube’ methods, rather than microscopic, though some fluorescent techniques have been developed. Betz and his co-workers have also published extensively on the immunohistochemistry of wound dating.
At the present time these methods still remain mainly in the realm of the research laboratory, apart from a few enthusiasts who employ them in casework. For the histo- chemical novice, it would be unwise to put such procedures into evidence without first gaining extensive experience of control material. Experience emphasizes that strictly standard conditions are required for the production of such histochemical sections, the end result depends largely on laboratory procedures – ‘change your technician and you change the answer’.
A list of standard procedures for some of these reactions is given in Appendix 1.
The following are some of the histochemical wound changes described by Raekallio and others:
■ In a wound through the skin surface there is a central zone 0.2–0.5 mm wide that will become necrotic and in which enzyme activity rapidly decreases. This may be termed ‘negative vital reaction’. Immediately beyond this layer, is a 0.1–0.3 mm zone of reaction and eventual repair, where a number of enzymes and other substances become increased in concentration during the reparative process, compared to the normal level in the areas outside the wound. Enzymatically, this can be called ‘positive vital reaction’, as no such zone develops in a post-mortem wound.
■ Within one hour after injury, esterases and adenosine triphosphatase increase in the positive zone. At around 2 hours, aminopeptidase activities increase and, at 4 hours, acid phosphatase activity increases. Alkaline phosphatase activity is delayed another hour or so though, of course, all these times are relative and subject to the usual biological variability.
■ Where death occurs at some stage of this process, the enzyme pattern is ‘frozen’ at that point and post-mortem changes do not substantially alter the reactions within a few days after death, especially if autolysis is kept at bay by refrigeration. Senility, severe illness and cachexia, as well as widespread multiple injuries, may distort the usual pattern by reducing the ability to produce these reparative enzymes. In contused wounds, the reactions are less useful than in cutaneous injuries, as the damage is more diffuse and there are no definite ‘zones’.
In all wounds, lack of reaction cannot be taken as indisputable proof of a post-mortem origin, though, if the opposite occurs, positive increase in the outer zone must only be the result of a vital reaction. Other enzymes have been used for wound dating: the review by Janssen (1984) in his authoritative book on forensic histology should be consulted for details, as well as the work of Betz.
■ Tissue cathepsins are said to increase almost immediately if the stroma is damaged, being demonstrable within 5–10 minutes. Two other substances are of use in establishing that wounds are ante-mortem and giving some idea as to their age. Both are produced during the inflammatory response that accompanies tissue damage – histamine and serotonin (5-hydroxytryptamine). They tend to be complementary to the enzymes, as they appear soon after infliction, within the hour or so before adenosine triphosphatase and esterases become detectable.
These vasoactive amines appear in maximum concentration about 10 minutes (serotonin) and 20–30 minutes (histamine) after wounding. One of the first demonstrations of this forensic aid was by Fazekas and Varagos-Kis (1965), who showed that a ligature mark in hanging revealed increased serotonin; presumably the hanging was atypical and the victim took 10 minutes to die which is rather unusual. For the employment of the test in human autopsy work, about 2 grams of skin freed from subcutaneous fat are sampled, together with a similar control sample from a nearby normal area of skin from the same corpse. This is to allow for the markedly different amounts found in different people and, indeed, in the same persons at different times.
To establish that a wound was ante-mortem rather than inflicted after death, the level of histamine in the wound must be at least 50 per cent greater than the control sample – and, for serotonin at least, twice the concentration of the control skin.