microscopy of bruises
History
The description of microscopic changes within sub-cutaneous haemorrhages dates back to Virchow, who described the presence of what was later identified as bilirubin pigment crystals and granular material associated with 'old' haemorrhagic areas in 1847, and Langhans who described the presence of phagocytosed red blood cells in haemorrhages in 1869 (Vanezis 2001).
Microscopic features of subcutaneous bruises
These have been reviewed by Vanezis 2001, and the earliest evidence of injury appears to be the formation of oedema in the vicinity of the haemorrhage, resulting in widening of fibrous septae.
Subsequent to the 'exudative' phase of healing, leucocyte infiltration is noted;
- Polymorph leucocytes have been noted to 'influx' into bruises after approximately 4 hours;
- mononuclear leukocytes after approximately 9 hours; and
- red blood cells appear in macrophages within 15 -17 hours of injury ( being present in some cases for several weeks) (Raekallio 1980).
Raekallio (1980) considered the presence of fibrin in sub-cutaneous haemorrhages and concluded that the clotting of tissue haemorrhage could occur post mortem - it was not evidence of a 'vital reaction', and the abscence of fibrin could not exclude 'vitality'.
However, post mortem fibrinolysis tended to occur within 1 day of the haemorrhage, and the finding of well-preserved fibrin networks in a sub-cutaneous haemorrhage examined at autopsy 2-3 days post mortem appeared to indicate a vital or agonal origin rather than post mortem origin.
Haemosiderin/ haemosiderin-laden macrophages
Haemosiderin-laden macrophages (detected by Perl's stain) can apparently be seen as early as 24-48 hours, but more commonly from 4-8 days. Bilirubin has been found in haemorrhages from 9 days.
However, in a morphometric study, the detection of 'considerable amounts of haemosiderin' (defined by Betz and Eisenmenger (1996) as 20% or more of the evaluated area), indicated a minimum wound age of approximately 1 week (although in the same study haemosiderin deposits were seen in a lesion 3 days old).
Haemosiderin formation was noted to depend on the extent of the initial haemorrhage and the 'physiological' reduction in the amount of this pigment with advanced wound age, and so slight or absent haemosiderin deposition could not provide information on a post-infliction interval.
Haemosiderin was found 'regularly' 90 hours post bruising and haematoidin 'occasionally' 9 days post bruising. Granulation tissue 'rarely' developed.
Perls-positive macrophages
Enzyme histochemistry
Enzyme-histochemistry showed an increase in ATPase activity in the vascular walls after 2.5 hours, whilst aminopeptidase and esterase activity increased at 4.5 and 7 hours respectively.
Immunohistochemistry
Nakajima and colleagues (2006) examined sub-cutaneous haemorrhages and observed heme oxygenase (HO)-1 expression and macrophage infiltration.
Heme oxygenase (HO) is the enzyme which catalyses the degradation of heme into biliverdin, carbon monoxide and iron, and this enzyme has been noted in phagocytic macrophages.
They found an increase in the HO-1 positive cells within 3 hours of injury, peaking at 3 days post-infliction. This increase corresponded to areas of macrophages, and they concluded that the determination of HO-1 expression might be useful for estimating the timne course of these injuries.
Apoptoses in bruises
Sawaguchi and colleagues (2000) studied apoptoses in bruised and normal skin using the TUNEL method of DNA end labelling. They found that the mean apoptotic cell count was statistically greater in bruised skin compared to normal skin in 2-6 day old bruises, and they suggested that there was a quiescent period prior to the increase in apoptotic cell activity in skin following bruising.
Animal studies
McCausland and Dougherty (1978) examined bruises in lambs and calves, and noted;
- lesions sampled immediately after bruising showed haemorrhage in subcutaneous tissues and between muscle fibres. Some polymorph neutrophils were present, together with macrophages and a fine fibrin network;
- the amount of haemorrhage was greater after 8 hours, and some muscle fibres showed degeneration;
- at 24 hours there was an equal ratio of polymorphs to macrophages, some of which contained haemosiderin; and
- at 48 hours there were more macrophages than polymorphs, and there were heavy deposits of haemosiderin and fibrin 'nets'.
references
- Nakajima T, Hayakawa M, Yajima D et al (2006), 'Time-course changes in the expression of heme oxygenase-1 in human subcutaneous haemorrhage', Forensic Science International 158:157-163
- Raekallio J (1980), 'Histological estimation of the age of injuries' (Ch.1) and 'Histochemical and biochemical estimation of the age of injuries' (Ch.2), In Perper JA and Wecht CH (Eds) 'Microscopic diagnosis in forensic pathology', Charles C Thomas, Springfield, Illinois
- Sawaguchi T, Jasani B, Kobayashi M, Knight B (2000), 'Post-mortem analysis of apoptotic changes associated with human skin bruises', Forensic Science International 108:187-203
- Vanezis P (2001), 'Bruising: Concepts of ageing and interpretation', Ch.11 In: 'Essentials of autopsy practice - Vol.1', Rutty GN (Ed), Springer-Verlag London Ltd