Forensic Science Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

DETERMINATION OF THE AGE OF WOUNDS BY HISTOCHEMICAL AND BIOCHEMICAL METHODS *

J. RAEKALLIO

Department of Forensic Medicine, University of Turku, 20520 Turku 52 (Finland)

SUMMARY

Methods of enzyme histochemistry can reveal vital changes around skin wounds and burns inflicted about one hour before death. This is about 8 times earlier than was possible before, when only histological methods were used. Histochemical methods act further as a guide to estimate the approximate age of wounds, especially of those inflicted l- 16 hours before death. However, conditions like senility, cachexia and severe brain injuries may impair the local reactions and thus complicate the dating of wounds.

The determination of the age of wounds becomes more reliable when several in- dependent methods are used simultaneously. The techniques of enzyme biochemistry seem to provide an experimental basis of new methods for the distinction between ante-mortem and post-mortem wounds. Isoelectric focusing in polyacrylamide gel has revealed qualitative differences between the enzymes (arylaminopeptidases and esterases) from ante-mortem and post-mortem wounds, respectively. Autopsy studies on the appli- cability of enzyme biochemical methods are in progress.

By contrast, biochemical histamine and serotonin determinations, as well as enzyme histochemistry, are applicable even in forensic practice. According to our autopsy studies, a distinct increase (twofold, at least, in regard to serotonin, and IS-fold or more, con- cerning histamine) in the serotonin and free histamine content, as compared to a control sample of neighbouring intact skin, indicates the ante-mortem origin of an injury. The increases in serotonin and histamine are the earliest vital reactions so far known, be- coming demonstrable after a survival time of as little as some minutes.

The distinction between ante-mortem and post-mortem injuries is one of the cardinal problems of forensic medicine. For example, a dead person may, after death, be run over by a car. If the forensic pathologist does not observe that the injuries were caused after death, innocent people may be arrested or even found guilty. On the other hand, lack of observations may lead to acquittal of guilty persons. For example, the dead body of a killed person may be put on railway lines in order to simulate an accident or a suicide.

* This paper was presented at the First South African International Symposium on Forensic Medi- cine, Johannesburg, South Africa, on September 3rd, 19 7 1.

Forens. Sci, 1 (1972) 3 - 16

4 J. RAEKALLIO

An ante-mortem wound is customarily identified by its profuse bleeding. However, Keith Simpson1 points out: “In any case in which it is doubtful whether a wound was inflicted on a living or dead body it would be most unwise to form an opinion on the extent or nature of haemorrhage, and no opinion should be given without microscopic sections being examined”.

Identification of a vital wound may, however, be impossible, even microscopically. Unfortunately, cellular infiltration (the most reliable histological criterion) becomes well marked only 4- 8 hours after wounding. This latent or “lag” phase is far too extensive to satisfy the demands of a medicolegal examination. On the other hand, reaction to injury must be immediate. The problem is, Are we able to detect the earliest reactions in a wound and thus demonstrate its ante-mortem or, in other words, vital, origin?

It is now knownze7 that morphological phenomena are preceded by functional changes and, moreover, that these correlate with the action of enzymes.

HISTOCHEMICAL METHODS

The histochemical methods have been described and discussed in detai13-5$7. For the histological and histochemical examination, the wounds (or parts of them) were excised with their surroundings, about half an inch in each direction. One half of the tissue block was fixed overnight in neutral 10% formalin at +4OC (ice-box temperature) for the demonstration of esterase and phosphatase activities and for histological examination. The other half was fresh-frozen using isopentane chilled with dry ice. The histochemical methods for adenosine triphosphatase and for (aryl)aminopeptidases were applied to the frozen specimen.

WOUND EDGE

CENTRAL ZONE

Fig. 1. Schematic diagram showing the zones of an ante-mortem skin wound.

AGE OF WOUNDS 5

Results and discussion of the histochemical studies

To summarize the results, 2 zones can be noticed around the vital wounds (Fig. 1). In the immediate vicinity of the wound edge, a central or superficial zone, 200-500~ deep, shows decreasing enzyme activity. This should be considered as an early sign of imminent necrosis. The regressive phenomena in the central zone may be called negative vital reactions since no such decrease in enzyme activity is observed in the wounds inflicted after death. The earliest histological signs of necrosis, karyolysis and karyorrhexis are demonstrable in &hour vital wounds, becoming more pronounced 16 hours after injury.

Surrounding the central area, a peripheral zone, 100-300~ deep,exhibits an increase in enzyme activity. The initial increase in enzyme activity represents, among other things, an adaptive defence mechanism of the local connective tissue cells as an enzymatic response to injury3p5,7. The increase in enzyme activity in the peripheral zone may be called a positive vital reaction since there are no such changes in post-mortem wounds. Figs. 2 and 3 are examples of this general pattern of enzyme activity in ante-mortem (vital) wounds.

The activity of esterases and adenosine triphosphatase increases as early as about one hour after injury; the activity of aminopeptidases increases at about 2 hours, and those of acid phosphatase and alkaline phosphatase at 4 and 8 hours, respectively. The consecutive appearance of the positive vital reactions, demonstrable by the various methods of enzyme histochemistry, allows the construction of a biological time-table which may be useful in the rough estimation of the age of vital wounds (Fig. 4).

Fig. 2. Aminopeptidase activity in a lacerated wound. Survival

removed one day after death.

Forens. Sci., 1 (1972) 3 - 16

time: 3 hours. The sample was

6 J. RAEKALLIO

Fig. 3. Aminopeptidase activity in a lacerated wound. The victim died 4 hours after wounding.

MononucIoarS Polymorpho -

nuclears Alkaline

phorphatase Acid

phorphatare Amino - peptidare

I Esterose

AT P-ose

0 1 2 4 6 8 16

AGE OF WOUNDS (Hours) Fig. 4. Schematic diagram showing the histochemical estimation of the age of ante-mortem skin wounds.

AGE OF WOUNDS 7

It is important, in practice, that the histochemical vital reactions are recognizable several days after death. There are, however, certain exceptions in the biological time- table when it is applied to human autopsy material. We collected material from 43 victims of traffic accidents. Most of the injuries were lacerated but there were also some incised wounds caused by glass or other sharp parts of the vehicles. The victims had survived for a known period of from some minutes to several days. The bodies were usually autopsied l-2 days after wounding. In this material there were 5 cases with an exceptional appearan- ce of enzymes (Table I). According to the data, conditions such as far-advanced senility, cachexia, very severe and multiple injuries, especially major brain injuries, may impair the local reaction of the skin. In this way such conditions could complicate the estimation of the age of skin wounds. Thus it is apparent that one ought never to date a wound on the basis of one histochemical or histological finding only. Now, as in the past, all autopsy findings, as well as the examination of the body at the scene and, when available, the case history, must all be taken into account.

TABLE I

THE EXCEPTIONAL CASES IN THE MATERIAL

Case Age (years)

Sex Survival time

Exceptions Cause of death

1 23 M 2 38 M 3 66 M 4 20 F 5 81 M

45 minutes E&erase +; ATPase * Spine fracture 90 minutes Esterase +; ATPase + Brain injury

3 hours* Aminopeptidase Brain injury 5 hours Acid phosphatase Bleeding

10 hours** Alkaline phosphatase Chest injury

* Cachexia (cancer) ** Senility

Histochemical findings, however, are often of great, sometimes even decisive, help to a forensic pathologist when he is faced with the problem of determining the age of wounds. The following example illustrates this: A young jealous man believed that he had killed his fiancBe when he hit her head with a stone. He carried the body onto railway lines in order to simulate an accident or a suicide. Four hours later a train arrived and caused fatal in- juries to the body. A medicolegal autopsy was performed and the wounds were studied using enzyme histochemical methods. According to the results, a laceration on her head was produced several hours before the fatal wounds caused by the train. The girl had ob- viously been unconscious after the hit on her head. The police were informed. The young man subsequently pleaded guilty when he realized that even the time-table of his crime was known.

Recently, some other investigators such as Lindne? and Pioch’ in Germany, Lo Menzo” (Italy), Fatteh’ ’ and Malik’ 2 (Northern Ireland), and Arimal 3 and Tana- ka14 in Japan, have studied vital and post-mortem skin wounds and burns using enzyme histochemistry. Their results are mainly compatible with mine.

Forens. Sci., 1 (1972) 3 - 16

8 J. RAEKALLIO

What new information have the methods of enzyme histochemistry provided? Firstly, they act as a guide to the approximate age of the wounds, especially those inflicted 1-16 hours before death. Secondly, the methods of enzyme histochemistry reveal vital changes around skin wounds, inflicted as little as one hour before death. This is about 8 times earlier than was possible before when only histological methods were used.

BIOCHEMICAL METHODS

I. Serotonin and histamine studies

Although enzyme histochemistry has shortened the latent period after wounding to about one eighth of that using conventional histology, there is still the very last hour before death, challenging further study. Histamine and serotonin (5hydroxytryptamine) are known to participate in the acute inflammatory process, especially in the earliest post-traumatic period. Fazekas and Viragos-Kis’ ’ observed, in 1965, an increase in the free histamine content in the ante-mortem ligature mark due to hanging. This observation prompted us to study the histamine content of skin wounds in controlled circumstances, i.e. initially on experimental animals . l6 We felt it important to study the other biogenic amine also, i.e. serotonin’ ’ , since the results certainly become more reliable when several independent methods are used simultaneously. In addition to experimental investigations, we studied histamine and serotonin in forensic autopsy material’ ‘.

A sample, about 2 g, of the injured skin, e.g. a piece of the ligature mark in cases of hanging, was removed. In addition, a control sample, equal in size, was taken from the neighbouring intact skin of the same body. This was necessary because there are great individual and regional differences in the free histamine and serotonin content of skin. Wounds inflicted at the autopsy served as additional controls. The extraction of the free histamine from skin was performed with Tyrode’s solution as described by Fazekas’ 5 I The free histamine of the extract was measured spectrofluorimetrically according to the method of Shore et al. lg. The serotonin was extracted and measured spectrofluorime- trically according to the method of Udenfriend et al. 20. For details of the methods the reader is referred to our previous publication’ ‘.

Results and discussion of the serotonin and histamine determinations

The acute increase in the free histamine content of guinea-pig skin is shown in Fig. 5. The maximal increase occurs within 20 - 30 minutes after wounding. The increase in the serotonin content is demonstrable still earlier, the maximal increase occurring within 10 minutes after wounding (Fig. 6). There is another, although slighter, increase in sero- tonin between 40 minutes and 2 hours.

The forensic autopsy material consisted of 20 bodies (Tables II and III). In 11 cases, both the serotonin and the free histamine content were determined; in 5 cases only the histamine content, and in 4 cases only the serotonin content, was measured. To demon-

AGE OF WOUNDS 9

2.0

1.0

01 I I I I I

0 10 20 30 40 50

WOUND AGE (MINUTES)

Fig. 5. Acute histamine reaction in the guinea-pig. The diagram shows relative free histamine contents of the wound surroundings, as compared to the free histamine values (=l) of a control sample of the same animal. The absolute values of the control samples of the free animals, depicted in this figure, were 4.9, 2.7 and 3.1 /Jg/g, respectively. In the figure, the curves represent the skin samples with the control values of 4.9, 2.7 and 3.1 ./_lg/g, respectively, in succession from above.

0 20 40 8 16 24

MINUTES HOURS WOUND AGE

Fig. 6. The diagram shows that the serotonin content depends on the age of vital wounds. The increase in the serotonin content in a group, wounded at a given time before death, is always com- pared to the serotonin content (=0 in the figure) of the post-mortem control wound of the same mice.

Forens. Sci., 1 (1972) 3 - 16

10 J. RAEKALLIO

strate this, the first 11 cases presented in Table III are numbered in duplicate, the second number (within parentheses) indicating the case number in Table II. The age and sex of the victims, the type of injury and its localization, its age (i.e. the time between the infliction of the injury and the death of the victim) are apparent from Tables II and III In most cases, the age of the injury was known. It was assumed that the victims of hanging had died, at most, within a few minutes. The post-mortem interval, ie. the time between death and autopsy, varied from 1 - 5 days (mean: 2.9 days).

In the skin injured intro vitum the free histamine values (Table II) were 1 .S - 5.5 times higher (mean: 2.4 times) than those of the respective control samples of the same indi- viduals. In one case (No. 5) the free histamine content did not increase and in 3 cases (Nos. 2, 7 and 11) it was less than 1.5 times higher than that of the control sample.

The vital increases in the serotonin content (Table III) were almost always manifold. In 4 cases (Nos. 2, 5, 6 and 9) the serotonin content of the skin injured intra vitam was about doubled; in all the cases the increase was pronounced, up to almost 30 times, the mean being an increase of 8.3 times.

There is no increase in the serotonin and free histamine content of post-mortem wounds. This is understandable since the release of both serotonin and histamine is an enzymatically regulated active process and needs energy. The energy required seems chiefly to be produced by oxidative phosphorylation. Death causes cessation of the blood flow and thus of oxygen transport. The lack of oxygen and of blood-borne oxidizable substances deprives the cells of the major source of energy for the synthesis of adenosine triphosphate. This uncouples oxidative phosphorylation, which is further hampered by the imminent malfunction of the cellular membranes, causing accumulation of calcium. This ion is a powerful inhibitor of oxidative phosphorylation. In spite of the uncoupling of oxidative phosphorylation many enzymes retain their ability to function, but even if they are still active the activity is no longer coordinated. This seems to apply, among others, to the oxidative enzymes degrading serotonin and histamine, and explains the fact that the increased amounts of serotonin and free histamine intra vitam are biochemically demonstrable several days after death.

The results of Berg et al 21 are compatible with ours. Determination of the histamine and serotonin content may play a decisive role in forensic practice. A farmer killed his wife by suffocation, stuffing her mouth and throat with cloth and other soft materials In order to simulate a suicide, he later hanged the dead body. He telephoned the police, telling the sad story of his wife’s suicide. As in most cases of suicide in Finland, a medicolegal autopsy was performed. No abrasions and only a few asphyxial petechiae were visible. A sample of the ligature mark and a control sample of the neighbouring uninjured skin were removed. In this case there were no distinct differences between the serotonin and histamine contents of the 2 samples, as is to be expected in a victim of suicidal hanging. The police were informed and the farmer subsequently pleaded guilty.

On the basis of the autopsy studies, a distinct increase in serotonin and free histamine content indicates the ante-mortem origin of an injury. (Distinct means an increase which is at least twofold in regard to serotonin, and 1.5fold or more for free histamine.) The

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AGE OF WOUNDS 13

increase in the serotonin content in the skin surrounding a wound indicates its vital origin even better than the increase in the free histamine content. This is because the free histamine content may decline even below the control level 30 - 40 minutes after experi- mental wounding. At that time it would thus be impossible to distinguish between vital and post-mortem wounds by the histamine method. The absence of the histamine reaction does not necessarily indicate that a wound was inflicted post mortem, but a positive histamine reaction, i.e. a distinct increase in the free histamine content, does indicate the vital origin of an injury. A distinct increase in the serotonin content also indicates the vital nature of a wound, and the increase in serotonin seems to be more manifest than that of free histamine.

2. Enzyme biochemical methods

It is obvious that the determination of the age of wounds becomes more reliable when several independent methods are used. Therefore, in addition to biochemical estimations of histamine and serotonin, and to enzyme histochemistry, we have applied enzyme biochemistry to the distinction between ante-mortem and post-mortem wounds.

We have previously shown biochemically that (aryl)aminopeptidase activity gradually increases in early wound healing, the intensification being greatest from 2 - 7 hours after vital injury* *. According to our studies, the modifier characteristics, as well as fractiona- tion by gel infiltration and by ion-exchange chromatography, show that the increased arylaminopeptidases in vital wounds differ qualitatively from the corresponding enzymes in intact skin or in post-mortem wounds23-2 5.

In order to detect further differences, we applied to (aryl)aminopeptidases the method of isoelectric focusing in polyacrylamide gel in order to distinguish between ante-mortem and post-mortem skin wounds. In our experimental study on rats, we tried to eliminate individual differences. For this purpose, the enzyme patterns of the ante-mortem lesions inflicted at different times were compared with those of wounds produced one hour after death in the same rats. The isoelectric focusing method used26 is based on the fact that qualitatively different (aryl)aminopeptidases distribute themselves in the polyacrylamide gel tubes according to the different isoelectric points of the respective enzymes. For details of the methods the reader is referred to our recent paper27.

Results and discussion of the enzyme biochemical studies

In the very earliest phase after wounding the (aryl)aminopeptidase activity was lower in the vital wounds than in the control wound preparations. This was observed clearly in the wounds inflicted 1.5 minutes before death and still seen in those made 30 minutes before death (Fig. 7). This decrease could result from inactivation of some arylamino- peptidases in the conditions (pH, for example) that changed during the wounding. Another explanation could be that some kind of leakage of the enzymes had taken place.

In vital wounds 30 minutes old (Fig. 7) however, an increase in activity or an ap-

Forens. Sci, 1 (1972) 3 -16

14 J. RAEKALLIC)

0,080

0,060

> 0,020 b 3 - 4 0

g 0,080 z = w 0,060

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0 0 10 20 30 LO 50 60

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Fig. 7. Isoelectric focusing of arylaminopeptidases in an ante-mortem wound (A) inflicted 30 minutes before death, and in a control (post-mortem) wound (B).

pearance of completely new enzymes was also seen. The activity of these new enzyme peaks further increased in wounds 1 hour old (Fig. 8). The increase of arylaminopeptidase activity cannot be explained as mere activation, since the new enzyme peaks, having different isoelectric points, represent different enzyme molecules from those present in the control tissue. Our results are compatible with those obtained by Jarecki et dz8. These authors studied another enzyme group, the esterases, using isoelectric focusing. The differences in esterases (as compared with post-mortem wounds) appeared within 30 minutes of vital injury.

The enzyme biochemical technique, isoeletric focusing in polyacrylamide gel, seems to provide an experimental basis for a new method for the biochemical distinction between ante-mortem and post-mortem wounds. Autopsy studies on the applicability of enzyme biochemical methods are in progress.

By contrast, enzyme histochemistry, and biochemical histamine and serotonin deter- minations, are also applicable to forensic practice.

AGE OF WOUNDS 15

0,160

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Fig. 8. Isoelectric focusing of arylaminopeptidases in an ante-mortem wound (A) inflicted one hour before death, and in a control (post-mortem) wound (B).

ACKNOWLEDGEMENT

This work was supported by research grants. from the Sigrid JusClius Foundation, Helsinki, and from the Finnish National Research Council for Medical Sciences.

REFERENCES

K. Simpson, Taylor’s Principles and Practice of Medical Jurisprudence, 12th ed., J. & A. Churchill, Ltd., London, 1965, p. 199. J. Raekallio, Enzymes histochemically demonstrable in the earliest phase of wound healing, Nature (Lond.), 188 (1960) 234 - 235. J. Raekallio, Histochemical studies on vital and post-mortem skin wounds: Experimental investi- gation on medicolegally significant vital reactions in an early phase of wound healing, Arm. Med. Exp. BioL Fenn. SuppI. 6, 39 (1961) 1 - 105. J. Raekalho, Histochemical distinction between antemortem and postmortem skin wounds, J. Forens. Sci., 9 (1964) 107 - 118. J. Raekallio, Die Alterbestimmung mechanisch bedingter Hautwunden mit enzymhistochemischen Methoden, Verlag Max Schmidt-RGmhild, Ltibeck, 1965.

Forens. Sci, 1 (1972) 3 - 16

16 J. RAEKALLIO

6 J. Raekallio, Enzyme histochemistry of vital and post-mortem skin wounds, J. Forens. Med., 13 (1966) 85 - 91.

7 J. Raekallio, Enzyme Histochemistry of Wound Healing, Gustav Fischer Verlag, Stuttgart, 1970. 8 J. Lindner, Vitale Reaktionen, Acta Histochem. (Jena), Supple 9, (1971) 435 - 467. 9 W. Pioch, Die histochemische Untersuchung thermischer Hautschiiden und ihre Bedeutung fiir die

forensische Praxis, Verlag Max Schmidt-Riimhild, Mbeck, 1966. 10 G. Lo Menzo, Lesione vitali e post-mortali, Edigraf, Catania,l966. 11 A. Fatteh, Histochemical distinction between antemortem and postmortem skin wounds, J.

Forens. Sci., 11 (1966) 17 ~ 27. 12 M.O.A. Malik, Histochemical changes as evidence of the antemortem origin of skin burns, J.

Forens. Sci, 15 (1970) 489 - 499. 13 T. Arima and H. Nagamori, A histochemical study on skin wound, Kagaku Keisatsu Kenkyosho

Hokoku, 18 (1965) 193 - 197. 14 M. Tanaka, The distinction between antemortem and postmortem skin wounds by esterase acti-

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