Forensic Science International 245 (2014) 72–76
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Fat from contused adipose tissue may cause yellow discoloration of clothes in blunt trauma victims D. Geisenberger a,*, F. Wuest b, L. Bielefeld a, M. Große Perdekamp a, R. Pircher a, S. Pollak a, A. Thierauf-Emberger a, L.M. Huppertz a a b
Institute of Forensic Medicine, Freiburg University Medical Centre, Freiburg, Germany Faculty of Biology, University of Freiburg, Freiburg, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 July 2014 Accepted 10 October 2014 Available online 18 October 2014
In some fatalities from intense blunt trauma, the victims’ clothes show strikingly yellow discoloration being in topographic correspondence with lacerated skin and crush damage to the underlying fatty tissue. This phenomenon is especially pronounced in light-colored textiles such as underwear made of cotton and in the absence of concomitant blood-staining. The constellation of findings seems to indicate that the fabric has been soaked with liquid body fat deriving from the contused adipose tissue. To check this hypothesis, textiles suspected to be contaminated with fat were investigated in 6 relevant cases. GC– MS-analysis proved the presence of 11 fatty acids. The fatty acid composition was similar to that of human adipose tissue with a high proportion of oleic acid (18:1). In total, the morphological and chemical findings demonstrated that the yellow discoloration of the victims’ clothes was caused by fat from traumatized adipose tissue. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Body fat Adipose tissue Blunt trauma Fatty acids Discoloration of textiles
1. Introduction The ability to think in terms of criminalistics has always been crucial for the medico-legal expert, since forensic medicine deals with the application of medical knowledge in the administration of justice [1,2]. Forensic autopsies fundamentally differ from autopsies performed by clinical pathologists [3,4] in that the investigation includes the inspection of the scene and the assessment of (biological) traces [5]. Therefore, the recognition and interpretation of trace material constitute an integral part of practical work aiming at the reconstruction of events. In this context, it has been repeatedly stressed that the forensic autopsy must not be confined to the ‘‘naked corpse’’ [6] as the decedent’s clothes may be a major source of evidence. The German guideline on forensic autopsy emphasizes the importance of investigating the clothing right at the beginning [7]. This requirement applies to all manners of death, i.e. homicides, suicides, accidents and fatalities from natural causes. Pedestrians killed in a car crash may serve as an example: Their garments are often stained with dust from the surface of the impacting vehicle,
* Corresponding author at: Institute of Forensic Medicine, Freiburg University Medical Centre, Albertstrasse 9, 79104 Freiburg, Germany. Tel.: +49 761 203 6831; fax: +49 761 203 6858. E-mail address: [email protected] (D. Geisenberger). http://dx.doi.org/10.1016/j.forsciint.2014.10.022 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
oily dirt from the car’s underbody, the imprint of a tire tread and traces of abraded or peeled off paint. Textiles can also be covered with adherent glass splinters, particles from the road surface or plant material from the shoulder. Last but not least, clothes are often soaked in fluids (e.g. water, blood, urine, petrol, engine oil and coolant). To the best of our knowledge, the pertinent literature does not mention body fat as a possible cause of textile staining in trauma victims. Several cases in point induced us to investigate this phenomenon more closely by analyzing the chemical composition of such fatty contaminations. 2. Material 2.1. Case 1 A 64-year-old male committed suicide and died from polytrauma with consecutive exsanguination after being hit by a rail vehicle from behind. Post-mortem lividity was sparse. The heart and aorta were ruptured. Both lower legs were severed. In the left axillary region, two linear tears ran toward the back of the body (Fig. 1). The skin between these two lesions was avulsed and the underlying fatty tissue was crushed. The left arm of the victim’s short-sleeved, white cotton undershirt showed intense yellow discoloration (Fig. 2).
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Fig. 1. (Belonging to case 1): Two linear lacerations in the left axillary and adjacent thoracic region with contused subcutis.
Fig. 2. (Belonging to case 1): Cotton undershirt stained with yellow fatty fluid in the area of the left sleeve.
2.2. Case 2
over by a truck in the region of the buttocks. The front of the right thigh showed an open de´collement. Postmortem lividity was sparse. A textile sample was taken from the victim’s beige cotton shorts partly discolored yellow. Subcutaneous tissue from the thigh region served as a reference sample.
A 69-year-old woman suffered a fatal polytrauma in a traffic accident as a pedestrian by being run over by a truck. Layerwise dissection of the soft tissues revealed an extensive de´collement on the entire back of the trunk. The heart was severed from the great vessels and the aorta was ruptured. Below the left buttock, a 13cm-long, deeply penetrating wound was located. A textile sample showing yellow discoloration was taken from the back of the victim’s white, long cotton underpants. A tear in the textile material corresponded to the impalement injury. Subcutaneous tissue from the gluteal region was used as a reference sample. 2.3. Case 3 A 51-year-old woman committed suicide by jumping into the railway track bed. The collision with a rail vehicle caused decapitation and transection of the upper part of the body with traumatic exposure of the thoracic and abdominal cavity accompanied by exenteration of thoracic and cervical organs. In the transitional zone between the thoracic and the abdominal region, the skin showed clean-cut circumferential severance. A textile sample was taken from the down-quilted waistcoat, which revealed yellow discoloration on the inside. Subcutaneous tissue from the preaxillary region served as reference sample. 2.4. Case 4 A 21-year-old woman suffered a fatal craniocerebral trauma with decerebration in a traffic accident as a cyclist. She was run
2.5. Case 5 A 48-year-old woman committed suicide on railway tracks. She was run over by a rail vehicle, which caused decapitation, rupture of the heart and aorta as well as amputation of the upper (Fig. 3) and lower extremities. Postmortem lividity was sparse. The superior thoracic aperture was exposed and the skin showed extensive avulsion with lacerations in the chest region. A textile sample was taken from a sleeve of the victim’s white cotton T-shirt, which was discolored yellow (Fig. 4). Subcutaneous tissue from the abdominal region was used as a reference sample. 2.6. Case 6 A 76-year-old female was killed in a collision with a passenger car. Death was caused by thoracic and abdominal trauma with pelvic fractures and ruptures of the left lung, the liver, the spleen and the right kidney. A subtotal traumatic amputation of the left upper arm was accompanied by extensive skin defects and severance of the left jugular vein. A textile sample was taken from the left side of the victim’s cotton undershirt, which was discolored yellow. Fatty tissue from the preaxillary region was used as a reference sample.
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D. Geisenberger et al. / Forensic Science International 245 (2014) 72–76
Fig. 4. (Belonging to case 5): White cotton undershirt showing extensive tearing and yellow staining (arrow ") together with minor blood soiling (arrow !) and patches of oily dirt (arrow ~; train collision).
Fig. 3. (Belonging to case 5): Subtotal traumatic amputation of the left arm in combination with crush damage of the subcutis.
hydroxide (TMSH). The samples were incubated for 10 min at 100 8C. For GC–MS analysis 1 ml was injected. 3.2. GC–MS analysis
3. Methods 3.1. Sample preparation Textile samples of about 5 cm2 suspected to be contaminated with fat were incubated for 1 h in 10 ml chloroform: methanol (2:1, v/v). After centrifugation (5 min, 3000 g), the supernatant was transferred to a new reaction tube and water was added up to 14 ml. Separation of phases was accelerated by centrifugation (5 min, 3000 g). The organic phase was then transferred to a new reaction tube and dried under a gentle flow of nitrogen. For saponification, the pellet was resuspended in 1.8 ml ethanol and 200 ml 10 N potassium hydroxide (KOH) and incubated for 30 min at 40 8C. For extraction of the fatty acids, the solution was acidified with 600 ml hydrochloric acid: water (1:4, v/v) and 2 ml petrolether (PE): diethylether (DE) (2:1, v/v) were added. After centrifugation (5 min, 3000 g), 100 ml of the organic phase were transferred to a new reaction tube and dried in a vacuum concentrator. Methylation was performed by resuspending the pellet in 100 ml methanol and adding 50 ml trimethylsulfonium
GC-MS analysis was performed using a Thermo Finnigan TraceGC coupled with a DSQII (Thermo Scientific) and electron impact (EI). Chromatographic separation was carried out on a TR-FAME column (30 m 0.25 mm 0.25 mm) from Thermo Scientific. The injector temperature was set to 260 8C and the injections were done in split mode with a split ratio of 10. Helium, at a constant flow of 1 ml/min, was used as carrier gas. The oven temperature was set to 100 8C, held for 2 min, then increased with 8 K/min to 250 8C and held for 2 min. Ion source temperature was 200 8C. Mass spectra were acquired from m/z 50 to 500. Compounds were identified by using the National Institute of Standards and Technology (NIST) database. 3.3. Data evaluation The peak areas of the different fatty acid methyl esters (FAMEs) were determined and the ratios of palmitic: palmitolic (16:0/16:1), oleic: stearic (18:1/18:0), and oleic: linoleic (18:1/18:2) acid methyl esters were calculated according to Haan et al. [8] and
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Fig. 5. Chromatogram of the five most dominant FAMEs in the adipose tissue sample of the first case. The distribution pattern is typical of adipose tissue.
Table 1 Quantitative ratios of three fatty acids in the discolored textiles (cases 1–6) and in the reference samples (adipose tissue from the victims). The ratios of the fatty acids in normal individuals and venous blood are taken from Schaaf et al. [10]. Fatty acid ratio C18:1/C18:0 Case
Textile sample
1 2 3 4 5 6 Adipose tissue in normal individuals Venous blood
17.45 23.89 4.28b 8.21 21.20 10.15
a b
Fatty acid ratio C18:1/C18:2 Reference sample (adipose tissue) a
31.91 12.17 8.80 20.56 10.26 11.9 3.9
Textile sample 6.74 6.01 4.18b 7.31 4.70 7.26
Reference sample (adipose tissue) a
5.35 7.30 6.36 4.28 6.83 3.7 1.0
No reference sample was taken during autopsy. The liquid fat was badly absorbed by the polyamide tissue of the down-quilted waistcoat worn by the victim.
Pollak et al. [9] (cf. Fig. 5). The values were compared to the distribution of fatty acids in human adipose tissue and blood [10]. 4. Results The 6 fatally injured victims constituting our study material underwent forensic autopsies. All of them had one characteristic in common, namely clothes soaked with yellow fatty fluid. The most important circumstances and autopsy findings have been outlined in chapter 2. In all cases, death had occurred already at the scene. 5 out of the 6 persons were females. The age at death was between 21 und 76 years (mean age 55). The body mass index (BMI) varied considerably (22 up to 30, mean 26). No victim was underweight, whereas 3 were either overweight or obese. Thus, persons with an elevated BMI were overrepresented in our study material. Three fatalities had to be classified as suicides (committed by intentionally inducing collision with a rail vehicle), the other 3 were due to traffic accidents (pedestrians struck and/or run over by a car or truck). In accordance with the kind and severity of the blunt traumatization, the injuries included ruptures of the heart and aorta or even subtotal evisceration. Therefore, immediate circulatory breakdown was to be assumed in each instance. At the site of impact, blunt force had caused extensive crush damage to the subcutaneous adipose tissue with consecutive release of liquid fat, which accumulated in pocket-like spaces under the skin. In the additional presence of a wound perforating or lacerating the integument, the emerging fat could steep the overlying garment, which thus turned yellow provided that it was a light-colored fabric and not blood-soaked. To test the hypothesis that the yellow fluid contaminating the clothes derived from traumatized adipose tissue, textile samples were subjected to an extraction procedure (chloroform–methanol). After saponification and subsequent methylation of the triglycerides present in the organic phase, 11 fatty acid methyl esters (FAMEs) could be identified by GC-MS. By comparing the peak areas, the proportions of some fatty acid methyl esters were
determined (Table 1). As a result, oleic acid was found to be prevailing both in the textile extracts and in the reference samples from subcutaneous tissue. Furthermore, FAME ratios were calculated yielding values characteristic for adipose tissue and not corresponding with the fatty acid composition in human plasma. 5. Discussion In blunt trauma victims, adipose tissue can be affected in many ways. The most common findings are subcutaneous hematomas originating from ruptured vessels. A special type of blunt injury is frequently seen in pedestrians struck or run over by motor or rail vehicles [11]: In such cases, the subcutaneous layer may be crushed and avulsed from the underlying fascia or bone forming a so-called pocket filled with fat released from disintegrated adipocytes. If the circulation continues for at least a short period, the pocket also contains more or less blood. In traumas followed by instantaneous circulatory breakdown, bleeding into the soft tissues and from skin wounds may be insignificant. A potential sequel to blunt trauma is pulmonary and systemic fat embolism, usually subsequent to bone fractures and/or damage of fatty tissue. Although there are different theories regarding the pathogenesis of fat embolism, most forensic pathologists agree that, at least in cases of physical trauma, fat globules from damaged adipocytes enter the lymphatic and/or venous blood vessels and lodge in the pulmonary capillaries [12,13]. In nontraumatic cases, the (rare) occurrence of fat embolism is explained by emulsion instability of the plasma lipids. Comparative analysis of adipose tissue and the pulmonary lipids in trauma victims with fat embolism yielded a similar fatty acid composition with a high proportion of the monounsaturated oleic acid (18:1). In healthy persons, the lipids in venous blood contain a much lower proportion of oleic acid comparable to that of linoleic acid (18:2). Therefore, the oleic acid: linoleic acid ratio can indicate whether the main proportion of triglycerides comes from the adipose tissue or from the blood [9].
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Our study material comprised 3 suicides (intentional railway collisions) and 3 traffic accidents (pedestrians struck and/or run over by vehicles). In all cases, death occurred already at the scene. The yellow textile discolorations were located in severely contused body regions. Besides, the skin of the affected area was lacerated so that the fat released from the damaged subcutis could issue and contaminate the overlying garment, mostly underwear. As the clothes were light-colored, the yellow staining was clearly discernible. Especially the clothes made of cotton were soaked with fatty fluid whereas synthetic textiles were contaminated to a lesser degree. The yellow coloration was not visible if the garment was additionally stained with blood. In our cases, the comparatively minor extent of external bleeding could be explained by the instantaneous circulatory collapse, mostly due to a rupture of the heart and/or the aorta. Of course the mere fact that a garment of a trauma victim (partially) changed to yellow does not yet prove that the discoloration was caused by body fat originating from damaged adipose tissue. Similar staining can arise from other contaminants such as urine, plasma, mineral oil or warning colors of pesticides [14]. Therefore, the chemical composition had to be clarified by analyzing samples of the affected clothes. By means of GC–MS 11 fatty acids could be determined. Compared to each other, oleic acid prevailed by far – quite similar to the proportions known in adipose tissue of humans. On the other hand, the fatty acid composition of the textile samples differed considerably from the pattern found in plasma where oleic acid is far less dominant than in the subcutis. Although there may be slight differences between body regions (e.g. buttocks vs. abdominal wall [15]), the prevalence of oleic acid is true for all subcutaneous sites. Accordingly, the oleic acid: linoleic acid ratio had shifted toward oleic acid in all textile samples independent of the injured body region. In this context, another two aspects deserve to be briefly mentioned. First, the fat of human adipose tissue is liquid in living persons and therefore pours out of a wound next to contused adipose tissue. This fact is often not considered as most autopsies are performed on refrigerated corpses. Second, the amount of liquid fat emerging from a wound at the site of impact depends, i.a., on the thickness of the bruised subcutaneous layer. Therefore, ceteris paribus a high body mass
index will be associated with a greater volume of fat that can contaminate the victim’s clothes. 6. Conclusions – In fatally injured victims of blunt trauma, light-colored garments may show a yellow discoloration originating from liquid body fat. – The fatty acid composition detected in the stained textiles corresponds to the fatty acid pattern found in adipose tissue. – A release of fat is only possible in body regions where the subcutaneous tissue has been extensively contused in association with a laceration of the skin. – Fat-induced yellow textile staining can be recognized best if the garment in question is not soaked with blood. References [1] J. Kratter, Lehrbuch der gerichtlichen Medizin, Enke, Stuttgart, 1912 p. 1. [2] S. Pollak, Medical criminalistics, Forensic Sci. Int. 165 (2007) 144–149. [3] P. Saukko, B. Knight, Knight’s Forensic Pathology, 3rd ed., Arnold, London, 2004, pp. 1–51. [4] P. Saukko, S. Pollak, Autopsy, in: J.A. Siegel, P.J. Saukko (Eds.), Encyclopedia of Forensic Sciences, 2nd ed., vol. 3, Academic Press, London, 2013, pp. 205–209. [5] W. Schwerd, Legal medicine and criminalistics, Z. Rechtsmed. 102 (1989) 421– 428. [6] O. Prokop, Forensische Medizin, 2nd ed., Volk und Gesundheit, Berlin, 1966 p. 214. [7] Deutsche Gesellschaft fu¨r Rechtsmedizin, Die rechtsmedizinische Leicheno¨ffnung, AWMF-Register 054/001, 2012, p. 1. [8] G.J. Haan, S. van der Heide, B.G. Wolthers, Analysis of fatty acids from human lipids by gaschromatography, J. Chromatogr. 162 (1979) 261–271. [9] S. Pollak, W. Vycudilik, C. Reiter, G. Remberg, W. Denk, Large droplet lipids in blood in the right ventricle, Z. Rechtsmed. 99 (1987) 109–119. [10] E. Schaaf, Die gaschromatographische Bestimmung der Fettsa¨uren des Depot-fett, des Knochenmarkes und des Serums. Ein Beitrag zur Untersuchung der Fettembolie, (Medical thesis), Univ of Heidelberg, 1972. [11] S. Pollak, A. Thierauf, Blunt force injury, in: B. Madea (Ed.), Handbook of Forensic Medicine, Wiley Blackwell, Chichester, 2014, pp. 258–282. [12] B. Brinkmann, M. Borgner, M. Bu¨low, Fat embolism of the lungs as the cause of death. Etiology, pathogenesis and reasoning, Z. Rechtsmed. 78 (1976) 255–272. [13] W. Holczabek, G. Machata, Gaschromatographische Untersuchung zur Frage der Entstehung der Fettembolie beim Menschen, Beitr. Gerichtl. Med. 28 (1971) 82–87. [14] A. Thierauf, W. Weinmann, V. Auwa¨rter, B. Vennemann, M. Bohnert, A survey of warning colours of pesticides, Forensic Sci. Med. Pathol. 6 (2010) 307–313. [15] G.T. Malcom, A.K. Bhattachargya, M. Velez-Duran, M.A. Guzman, M.C. Oalmann, J.P. Strong, Fatty acid composition of adipose tissue in humans: differences between subcutaneous sites, Am. J. Clin. Nutr. 50 (1989) 288–291.
Contents lists available at ScienceDirect
Forensic Science International journal homepage: www.elsevier.com/locate/forsciint
Fat from contused adipose tissue may cause yellow discoloration of clothes in blunt trauma victims D. Geisenberger a,*, F. Wuest b, L. Bielefeld a, M. Große Perdekamp a, R. Pircher a, S. Pollak a, A. Thierauf-Emberger a, L.M. Huppertz a a b
Institute of Forensic Medicine, Freiburg University Medical Centre, Freiburg, Germany Faculty of Biology, University of Freiburg, Freiburg, Germany
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 July 2014 Accepted 10 October 2014 Available online 18 October 2014
In some fatalities from intense blunt trauma, the victims’ clothes show strikingly yellow discoloration being in topographic correspondence with lacerated skin and crush damage to the underlying fatty tissue. This phenomenon is especially pronounced in light-colored textiles such as underwear made of cotton and in the absence of concomitant blood-staining. The constellation of findings seems to indicate that the fabric has been soaked with liquid body fat deriving from the contused adipose tissue. To check this hypothesis, textiles suspected to be contaminated with fat were investigated in 6 relevant cases. GC– MS-analysis proved the presence of 11 fatty acids. The fatty acid composition was similar to that of human adipose tissue with a high proportion of oleic acid (18:1). In total, the morphological and chemical findings demonstrated that the yellow discoloration of the victims’ clothes was caused by fat from traumatized adipose tissue. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Body fat Adipose tissue Blunt trauma Fatty acids Discoloration of textiles
1. Introduction The ability to think in terms of criminalistics has always been crucial for the medico-legal expert, since forensic medicine deals with the application of medical knowledge in the administration of justice [1,2]. Forensic autopsies fundamentally differ from autopsies performed by clinical pathologists [3,4] in that the investigation includes the inspection of the scene and the assessment of (biological) traces [5]. Therefore, the recognition and interpretation of trace material constitute an integral part of practical work aiming at the reconstruction of events. In this context, it has been repeatedly stressed that the forensic autopsy must not be confined to the ‘‘naked corpse’’ [6] as the decedent’s clothes may be a major source of evidence. The German guideline on forensic autopsy emphasizes the importance of investigating the clothing right at the beginning [7]. This requirement applies to all manners of death, i.e. homicides, suicides, accidents and fatalities from natural causes. Pedestrians killed in a car crash may serve as an example: Their garments are often stained with dust from the surface of the impacting vehicle,
* Corresponding author at: Institute of Forensic Medicine, Freiburg University Medical Centre, Albertstrasse 9, 79104 Freiburg, Germany. Tel.: +49 761 203 6831; fax: +49 761 203 6858. E-mail address: [email protected] (D. Geisenberger). http://dx.doi.org/10.1016/j.forsciint.2014.10.022 0379-0738/ß 2014 Elsevier Ireland Ltd. All rights reserved.
oily dirt from the car’s underbody, the imprint of a tire tread and traces of abraded or peeled off paint. Textiles can also be covered with adherent glass splinters, particles from the road surface or plant material from the shoulder. Last but not least, clothes are often soaked in fluids (e.g. water, blood, urine, petrol, engine oil and coolant). To the best of our knowledge, the pertinent literature does not mention body fat as a possible cause of textile staining in trauma victims. Several cases in point induced us to investigate this phenomenon more closely by analyzing the chemical composition of such fatty contaminations. 2. Material 2.1. Case 1 A 64-year-old male committed suicide and died from polytrauma with consecutive exsanguination after being hit by a rail vehicle from behind. Post-mortem lividity was sparse. The heart and aorta were ruptured. Both lower legs were severed. In the left axillary region, two linear tears ran toward the back of the body (Fig. 1). The skin between these two lesions was avulsed and the underlying fatty tissue was crushed. The left arm of the victim’s short-sleeved, white cotton undershirt showed intense yellow discoloration (Fig. 2).
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Fig. 1. (Belonging to case 1): Two linear lacerations in the left axillary and adjacent thoracic region with contused subcutis.
Fig. 2. (Belonging to case 1): Cotton undershirt stained with yellow fatty fluid in the area of the left sleeve.
2.2. Case 2
over by a truck in the region of the buttocks. The front of the right thigh showed an open de´collement. Postmortem lividity was sparse. A textile sample was taken from the victim’s beige cotton shorts partly discolored yellow. Subcutaneous tissue from the thigh region served as a reference sample.
A 69-year-old woman suffered a fatal polytrauma in a traffic accident as a pedestrian by being run over by a truck. Layerwise dissection of the soft tissues revealed an extensive de´collement on the entire back of the trunk. The heart was severed from the great vessels and the aorta was ruptured. Below the left buttock, a 13cm-long, deeply penetrating wound was located. A textile sample showing yellow discoloration was taken from the back of the victim’s white, long cotton underpants. A tear in the textile material corresponded to the impalement injury. Subcutaneous tissue from the gluteal region was used as a reference sample. 2.3. Case 3 A 51-year-old woman committed suicide by jumping into the railway track bed. The collision with a rail vehicle caused decapitation and transection of the upper part of the body with traumatic exposure of the thoracic and abdominal cavity accompanied by exenteration of thoracic and cervical organs. In the transitional zone between the thoracic and the abdominal region, the skin showed clean-cut circumferential severance. A textile sample was taken from the down-quilted waistcoat, which revealed yellow discoloration on the inside. Subcutaneous tissue from the preaxillary region served as reference sample. 2.4. Case 4 A 21-year-old woman suffered a fatal craniocerebral trauma with decerebration in a traffic accident as a cyclist. She was run
2.5. Case 5 A 48-year-old woman committed suicide on railway tracks. She was run over by a rail vehicle, which caused decapitation, rupture of the heart and aorta as well as amputation of the upper (Fig. 3) and lower extremities. Postmortem lividity was sparse. The superior thoracic aperture was exposed and the skin showed extensive avulsion with lacerations in the chest region. A textile sample was taken from a sleeve of the victim’s white cotton T-shirt, which was discolored yellow (Fig. 4). Subcutaneous tissue from the abdominal region was used as a reference sample. 2.6. Case 6 A 76-year-old female was killed in a collision with a passenger car. Death was caused by thoracic and abdominal trauma with pelvic fractures and ruptures of the left lung, the liver, the spleen and the right kidney. A subtotal traumatic amputation of the left upper arm was accompanied by extensive skin defects and severance of the left jugular vein. A textile sample was taken from the left side of the victim’s cotton undershirt, which was discolored yellow. Fatty tissue from the preaxillary region was used as a reference sample.
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Fig. 4. (Belonging to case 5): White cotton undershirt showing extensive tearing and yellow staining (arrow ") together with minor blood soiling (arrow !) and patches of oily dirt (arrow ~; train collision).
Fig. 3. (Belonging to case 5): Subtotal traumatic amputation of the left arm in combination with crush damage of the subcutis.
hydroxide (TMSH). The samples were incubated for 10 min at 100 8C. For GC–MS analysis 1 ml was injected. 3.2. GC–MS analysis
3. Methods 3.1. Sample preparation Textile samples of about 5 cm2 suspected to be contaminated with fat were incubated for 1 h in 10 ml chloroform: methanol (2:1, v/v). After centrifugation (5 min, 3000 g), the supernatant was transferred to a new reaction tube and water was added up to 14 ml. Separation of phases was accelerated by centrifugation (5 min, 3000 g). The organic phase was then transferred to a new reaction tube and dried under a gentle flow of nitrogen. For saponification, the pellet was resuspended in 1.8 ml ethanol and 200 ml 10 N potassium hydroxide (KOH) and incubated for 30 min at 40 8C. For extraction of the fatty acids, the solution was acidified with 600 ml hydrochloric acid: water (1:4, v/v) and 2 ml petrolether (PE): diethylether (DE) (2:1, v/v) were added. After centrifugation (5 min, 3000 g), 100 ml of the organic phase were transferred to a new reaction tube and dried in a vacuum concentrator. Methylation was performed by resuspending the pellet in 100 ml methanol and adding 50 ml trimethylsulfonium
GC-MS analysis was performed using a Thermo Finnigan TraceGC coupled with a DSQII (Thermo Scientific) and electron impact (EI). Chromatographic separation was carried out on a TR-FAME column (30 m 0.25 mm 0.25 mm) from Thermo Scientific. The injector temperature was set to 260 8C and the injections were done in split mode with a split ratio of 10. Helium, at a constant flow of 1 ml/min, was used as carrier gas. The oven temperature was set to 100 8C, held for 2 min, then increased with 8 K/min to 250 8C and held for 2 min. Ion source temperature was 200 8C. Mass spectra were acquired from m/z 50 to 500. Compounds were identified by using the National Institute of Standards and Technology (NIST) database. 3.3. Data evaluation The peak areas of the different fatty acid methyl esters (FAMEs) were determined and the ratios of palmitic: palmitolic (16:0/16:1), oleic: stearic (18:1/18:0), and oleic: linoleic (18:1/18:2) acid methyl esters were calculated according to Haan et al. [8] and
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Fig. 5. Chromatogram of the five most dominant FAMEs in the adipose tissue sample of the first case. The distribution pattern is typical of adipose tissue.
Table 1 Quantitative ratios of three fatty acids in the discolored textiles (cases 1–6) and in the reference samples (adipose tissue from the victims). The ratios of the fatty acids in normal individuals and venous blood are taken from Schaaf et al. [10]. Fatty acid ratio C18:1/C18:0 Case
Textile sample
1 2 3 4 5 6 Adipose tissue in normal individuals Venous blood
17.45 23.89 4.28b 8.21 21.20 10.15
a b
Fatty acid ratio C18:1/C18:2 Reference sample (adipose tissue) a
31.91 12.17 8.80 20.56 10.26 11.9 3.9
Textile sample 6.74 6.01 4.18b 7.31 4.70 7.26
Reference sample (adipose tissue) a
5.35 7.30 6.36 4.28 6.83 3.7 1.0
No reference sample was taken during autopsy. The liquid fat was badly absorbed by the polyamide tissue of the down-quilted waistcoat worn by the victim.
Pollak et al. [9] (cf. Fig. 5). The values were compared to the distribution of fatty acids in human adipose tissue and blood [10]. 4. Results The 6 fatally injured victims constituting our study material underwent forensic autopsies. All of them had one characteristic in common, namely clothes soaked with yellow fatty fluid. The most important circumstances and autopsy findings have been outlined in chapter 2. In all cases, death had occurred already at the scene. 5 out of the 6 persons were females. The age at death was between 21 und 76 years (mean age 55). The body mass index (BMI) varied considerably (22 up to 30, mean 26). No victim was underweight, whereas 3 were either overweight or obese. Thus, persons with an elevated BMI were overrepresented in our study material. Three fatalities had to be classified as suicides (committed by intentionally inducing collision with a rail vehicle), the other 3 were due to traffic accidents (pedestrians struck and/or run over by a car or truck). In accordance with the kind and severity of the blunt traumatization, the injuries included ruptures of the heart and aorta or even subtotal evisceration. Therefore, immediate circulatory breakdown was to be assumed in each instance. At the site of impact, blunt force had caused extensive crush damage to the subcutaneous adipose tissue with consecutive release of liquid fat, which accumulated in pocket-like spaces under the skin. In the additional presence of a wound perforating or lacerating the integument, the emerging fat could steep the overlying garment, which thus turned yellow provided that it was a light-colored fabric and not blood-soaked. To test the hypothesis that the yellow fluid contaminating the clothes derived from traumatized adipose tissue, textile samples were subjected to an extraction procedure (chloroform–methanol). After saponification and subsequent methylation of the triglycerides present in the organic phase, 11 fatty acid methyl esters (FAMEs) could be identified by GC-MS. By comparing the peak areas, the proportions of some fatty acid methyl esters were
determined (Table 1). As a result, oleic acid was found to be prevailing both in the textile extracts and in the reference samples from subcutaneous tissue. Furthermore, FAME ratios were calculated yielding values characteristic for adipose tissue and not corresponding with the fatty acid composition in human plasma. 5. Discussion In blunt trauma victims, adipose tissue can be affected in many ways. The most common findings are subcutaneous hematomas originating from ruptured vessels. A special type of blunt injury is frequently seen in pedestrians struck or run over by motor or rail vehicles [11]: In such cases, the subcutaneous layer may be crushed and avulsed from the underlying fascia or bone forming a so-called pocket filled with fat released from disintegrated adipocytes. If the circulation continues for at least a short period, the pocket also contains more or less blood. In traumas followed by instantaneous circulatory breakdown, bleeding into the soft tissues and from skin wounds may be insignificant. A potential sequel to blunt trauma is pulmonary and systemic fat embolism, usually subsequent to bone fractures and/or damage of fatty tissue. Although there are different theories regarding the pathogenesis of fat embolism, most forensic pathologists agree that, at least in cases of physical trauma, fat globules from damaged adipocytes enter the lymphatic and/or venous blood vessels and lodge in the pulmonary capillaries [12,13]. In nontraumatic cases, the (rare) occurrence of fat embolism is explained by emulsion instability of the plasma lipids. Comparative analysis of adipose tissue and the pulmonary lipids in trauma victims with fat embolism yielded a similar fatty acid composition with a high proportion of the monounsaturated oleic acid (18:1). In healthy persons, the lipids in venous blood contain a much lower proportion of oleic acid comparable to that of linoleic acid (18:2). Therefore, the oleic acid: linoleic acid ratio can indicate whether the main proportion of triglycerides comes from the adipose tissue or from the blood [9].
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Our study material comprised 3 suicides (intentional railway collisions) and 3 traffic accidents (pedestrians struck and/or run over by vehicles). In all cases, death occurred already at the scene. The yellow textile discolorations were located in severely contused body regions. Besides, the skin of the affected area was lacerated so that the fat released from the damaged subcutis could issue and contaminate the overlying garment, mostly underwear. As the clothes were light-colored, the yellow staining was clearly discernible. Especially the clothes made of cotton were soaked with fatty fluid whereas synthetic textiles were contaminated to a lesser degree. The yellow coloration was not visible if the garment was additionally stained with blood. In our cases, the comparatively minor extent of external bleeding could be explained by the instantaneous circulatory collapse, mostly due to a rupture of the heart and/or the aorta. Of course the mere fact that a garment of a trauma victim (partially) changed to yellow does not yet prove that the discoloration was caused by body fat originating from damaged adipose tissue. Similar staining can arise from other contaminants such as urine, plasma, mineral oil or warning colors of pesticides [14]. Therefore, the chemical composition had to be clarified by analyzing samples of the affected clothes. By means of GC–MS 11 fatty acids could be determined. Compared to each other, oleic acid prevailed by far – quite similar to the proportions known in adipose tissue of humans. On the other hand, the fatty acid composition of the textile samples differed considerably from the pattern found in plasma where oleic acid is far less dominant than in the subcutis. Although there may be slight differences between body regions (e.g. buttocks vs. abdominal wall [15]), the prevalence of oleic acid is true for all subcutaneous sites. Accordingly, the oleic acid: linoleic acid ratio had shifted toward oleic acid in all textile samples independent of the injured body region. In this context, another two aspects deserve to be briefly mentioned. First, the fat of human adipose tissue is liquid in living persons and therefore pours out of a wound next to contused adipose tissue. This fact is often not considered as most autopsies are performed on refrigerated corpses. Second, the amount of liquid fat emerging from a wound at the site of impact depends, i.a., on the thickness of the bruised subcutaneous layer. Therefore, ceteris paribus a high body mass
index will be associated with a greater volume of fat that can contaminate the victim’s clothes. 6. Conclusions – In fatally injured victims of blunt trauma, light-colored garments may show a yellow discoloration originating from liquid body fat. – The fatty acid composition detected in the stained textiles corresponds to the fatty acid pattern found in adipose tissue. – A release of fat is only possible in body regions where the subcutaneous tissue has been extensively contused in association with a laceration of the skin. – Fat-induced yellow textile staining can be recognized best if the garment in question is not soaked with blood. References [1] J. Kratter, Lehrbuch der gerichtlichen Medizin, Enke, Stuttgart, 1912 p. 1. [2] S. Pollak, Medical criminalistics, Forensic Sci. Int. 165 (2007) 144–149. [3] P. Saukko, B. Knight, Knight’s Forensic Pathology, 3rd ed., Arnold, London, 2004, pp. 1–51. [4] P. Saukko, S. Pollak, Autopsy, in: J.A. Siegel, P.J. Saukko (Eds.), Encyclopedia of Forensic Sciences, 2nd ed., vol. 3, Academic Press, London, 2013, pp. 205–209. [5] W. Schwerd, Legal medicine and criminalistics, Z. Rechtsmed. 102 (1989) 421– 428. [6] O. Prokop, Forensische Medizin, 2nd ed., Volk und Gesundheit, Berlin, 1966 p. 214. [7] Deutsche Gesellschaft fu¨r Rechtsmedizin, Die rechtsmedizinische Leicheno¨ffnung, AWMF-Register 054/001, 2012, p. 1. [8] G.J. Haan, S. van der Heide, B.G. Wolthers, Analysis of fatty acids from human lipids by gaschromatography, J. Chromatogr. 162 (1979) 261–271. [9] S. Pollak, W. Vycudilik, C. Reiter, G. Remberg, W. Denk, Large droplet lipids in blood in the right ventricle, Z. Rechtsmed. 99 (1987) 109–119. [10] E. Schaaf, Die gaschromatographische Bestimmung der Fettsa¨uren des Depot-fett, des Knochenmarkes und des Serums. Ein Beitrag zur Untersuchung der Fettembolie, (Medical thesis), Univ of Heidelberg, 1972. [11] S. Pollak, A. Thierauf, Blunt force injury, in: B. Madea (Ed.), Handbook of Forensic Medicine, Wiley Blackwell, Chichester, 2014, pp. 258–282. [12] B. Brinkmann, M. Borgner, M. Bu¨low, Fat embolism of the lungs as the cause of death. Etiology, pathogenesis and reasoning, Z. Rechtsmed. 78 (1976) 255–272. [13] W. Holczabek, G. Machata, Gaschromatographische Untersuchung zur Frage der Entstehung der Fettembolie beim Menschen, Beitr. Gerichtl. Med. 28 (1971) 82–87. [14] A. Thierauf, W. Weinmann, V. Auwa¨rter, B. Vennemann, M. Bohnert, A survey of warning colours of pesticides, Forensic Sci. Med. Pathol. 6 (2010) 307–313. [15] G.T. Malcom, A.K. Bhattachargya, M. Velez-Duran, M.A. Guzman, M.C. Oalmann, J.P. Strong, Fatty acid composition of adipose tissue in humans: differences between subcutaneous sites, Am. J. Clin. Nutr. 50 (1989) 288–291.
