Sudden death after exercise in an adolescent with hemoglobin SE

Hemoglobin (Hb) S and Hb E are the most common variant hemoglobins, but because of the geographical separation of the areas where they are prevalent, the combination of the 2 is uncommon. Approximately 46 cases of hemoglobin SE compound heterozygosity have been reported. No deaths from the condition have been reported previously, whereas death after vigorous physical activity in individuals with sickle cell trait (hemoglobin AS) has been described in a few case reports. Here we report previously undiagnosed hemoglobinopathy SE in a 12-year-old American boy who collapsed during football practice and had a cardiac arrest on the field after a brief lucid interval. The autopsy was significant only for postmortem intravascular sickling. A postmortem hemoglobin electrophoresis test revealed 57% S, 34% E, and 1% F hemoglobins. The death is attributed to cardiac ischemia from functional vaso-occlusion by sickled erythrocytes.     

Hypostasis-induced changes in the breast area

When the anterior thoracic wall is within the hypostatic area, postmortem lividity in the region of the nipples and their immediate vicinity is less intense or absent. Where hypostatic skin haemorrhages (so-called vibices) are present, these are also found outside the areola of the nipple only. This phenomenon is visible not only externally, but is particularly pronounced on the cut surface. A possible explanation may be the different distribution pattern of connective tissue, smooth muscles and blood vessels inside and outside the areola mammae. Based on 13 cases from the forensic autopsy material--mostly drug related deaths--the macromorphological findings and their histological correlates are presented.     

Immunohistochemical detection of fibronectin and tenascin in incised human skin injuries

Immunohistochemical detection of molecules involved in inflammatory reaction can be useful for the diagnosis of vitality in skin wounds. We studied the expression of fibronectin (FN) and tenascin (TN) in 58 human skin wounds (48 vital and 10 postmortem). The age of vital injuries ranged from 3 min to 8 h and postmortem specimens were collected after a postinfliction interval of 15-180 min. One hundred thirty-seven formalin-fixed paraffin-embedded sections (mean: 2.3 sections per case) were stained with each of two monoclonal antibodies against FN and TN using the streptABC technique. A reticular staining for FN in wound edge and dermis was observed in 50% of vital specimens versus 0% in postmortem cases. Immunoreactivity was reduced in 10 autolysed cases. FN positivity exclusively at the injury margin was observed in 39.4% of vital wounds and 10% of postmortem cases. TN was negative in all specimens. Vital and postmortem hemorrhage areas showed positivity for FN and TN. Due to its low sensitivity, immunohistochemical analysis of FN is useful for determining vitality only in a minority of cases. Different factors in everyday practice, including autolysis and technical problems often produce false negative reactions with the result that FN cannot be regarded as a reliable parameter of vitality. Positive reactions (network staining) are more valuable than negativity but are not pathognomonic. Both vital and postmortem hemorrhages show an enhanced positivity for FN and TN, thus impeding the diagnosis.     

Distinguishing antemortem from postmortem injuries by LTB4 quantification

Leukotriene B₄ (LTB₄) in skin samples from seven forensic cases was detected by HPLC to distinguish their antemortem or postmortem origin. In total, there were thirteen antemortem and seven postmortem specimens. The results showed that LTB₄ was found in all antemortem wound specimens which were either fresh, or refrigerated or fixed in formalin for less than 10 days. In contrast, LTB₄ could not be detected in postmortem wound specimens. These results suggested that detecting the content of LTB₄ is a useful method for distinguishing antemortem from postmortem injuries.     

Pink teeth in a series of bodies recovered from a single shipwreck

Pink teeth have most often been observed in victims of drowning but have also been reported in subjects who died suddenly and unnaturally. There is general agreement that there is no obvious connection between the occurrence of pink teeth and the cause of death, but the condition of the surroundings (especially humidity) must certainly play an important role in the development of the pink-tooth phenomenon. The frequency and distribution of postmortem pink coloration of the teeth have been studied among a representative sample of 52 cadavers. All the bodies were victims of a single shipwreck that occurred on March 13, 1997, in the middle of the Otranto Canal (Mediterranean Sea). The bodies were recovered from the seawater after approximately 7 months. A distinct pink coloration of the teeth was found in only 18 cadavers (13 females and 5 males) of ages ranging between 13 and 60 years. The phenomenon was more pronounced in younger individuals due to age-related changes of the root canal, less penetrable by the pigment responsible for the postmortem pink staining. By histochemical methods and autofluorescence, hemoglobin and its derivatives have been identified as the most likely pigments responsible for this postmortem process that can be considered analogous to postmortem lividity. These data are consistent with previous reports on pink teeth, indicating that the diffusion of the blood in the pulp into the dentinal tubules causes the red discoloration of the teeth. Based on the results, the pigmentation is more prominent on the teeth with single roots rather than in the posterior teeth with multiple roots.     

Postmortem changes mistaken for traumatic lesions: a highly prevalent reason for coroner's autopsy request

Postmortem changes are well known for their possible misinterpretation as traumatic lesions which can mislead to suspicion of violent death and therefore to a forensic autopsy request. As far as we know, a systematic review of the prevalence of such a reason for coroner's autopsy request has not been done yet. A retrospective study of 230 forensic autopsies requested by the Coroner's office from 2002 to 2004 in the province of Quebec, Canada, was conducted by the authors. Of the 230 reviewed cases, postmortem artifacts mistaken for traumatic lesions were found in 18 cases. These misinterpretation were based on 5 categories of portmortem changes: purge fluid drainage in 12 cases (66.7%), bluish discoloration by lividity in 5 cases (27.8%), parchment-like drying of the skin in 4 cases (22.2%), bloating from gas formation in 4 cases (22.2%), and skin slippage in 1 case (5.56%). Therefore, postmortem artifacts misinterpretation occurred in 7.83% (95% confidence interval 0.05-0.12) of all requested forensic autopsies and in 35.29% (95% confidence interval 0.23-0.50) of decomposed autopsy cases. This study clearly establishes the high prevalence of postmortem artifacts as main reason for forensic autopsy request. Hence, in a context of forensic pathologist shortage, the improvement of coroner continuous training may reduce the workload.     

Factors and circumstances influencing the development of hemorrhages in livor mortis

Petechial hemorrhages or ecchymoses in the skin of the face and/or in eyelids and/or conjunctivae are one important feature in postmortem diagnosis of lethal strangulation. On the other hand, petechial bleedings can occur in various causes of death, especially in cases of neck or thoracic compression, they can occur in acute cardiac failure, as a result of blood or skin diseases or as a postmortem phenomenon. The focus of this investigation (retrospective study of 279 corpses, found initially in a prone position or some other face down position) was to analyse the frequency of postmortem (hypostatic) hemorrhages and factors which may influence their development. Petechial hemorrhages in livor mortis in the skin of the trunk and extremities were found in 110 cases (39%). The frequency ranged from 41% in the side position and 44% in the kneeling position to 50% in the prone position. Increasing intensity of livor mortis resulted in an increasing frequency of hemorrhages, up to 59%. In cases with a body-mass-index (BMI) of more than 26 the frequency of hemorrhages increased up to 64%. In cases without livor mortis when the corpses were found as well as in cases with complete movement of livor mortis after turning the corpses, no hemorrhages were found. If hypostasis was partly or completely fixed, the rate of hemorrhages increased up to 50%, without additional increase in longer postmortem intervals. Obviously postmortem petechiae develop neither very soon nor days after death, but within a period of several hours after death.     

Time-dependent immunohistochemical detection of proinflammatory cytokines (IL-1beta,IL-6, TNF-alpha) in human skin wounds

The proinflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) hold important functions in the early and late courses of inflammation, trauma and wound healing. In the present study, human skin wounds due to sharp force (n = 105) were collected during surgery and autopsy. The wound age mainly varied from several minutes to 5 h, some specimens aged up to 6 weeks. Control specimens from uninjured skin were available in each case. After preparation of cryostat sections, immunohistochemistry was performed according to the APAAP technique, using monoclonal and polyclonal antibodies. The results were evaluated semiquantitatively. All markers were weakly expressed in normal human skin constitutively. However, the staining pattern changed significantly in vital wounds concerning epidermal layers, subepidermal cells, vessels and sweat glands. IL-1beta and IL-6 showed enhanced expression after 15 and 20 min at the earliest (increase of epidermal reactivity). After 30-60 and 60-90 min, respectively, marked expression was observed with these markers. Similar alterations were detectable with TNF-alpha after 15 and 60-90 min. The reactivity of all three markers persisted over several hours, then decreased to basal levels again and sometimes reappeared after days and in granulation tissue. Leukocytes reacting with IL-1beta and IL-6 appeared after approximately 2 h. CONCLUSION: proinflammatory cytokines can serve as a useful tool for the estimation of vitality and wound age, in particular in the early post-traumatic interval prior to leukocyte reaction. Autolysis did not play a role in the samples investigated (postmortem interval up to 8 days). Problems could sometimes rise from constitutive expression. Therefore, it is recommended to examine control samples from the same individual and to compare the reactivity with wound specimens.     

Postmortem pink teeth.

A series of cases is reported in which pink teeth were observed during the postmortem period. Most cases were associated with decomposition in a moist environment. Experimental procedures led to the extraction of pink material from dentin and demonstration that hemoglobin and serum proteins were present. The pink-tooth phenomenon was duplicated in human teeth by instilling into the pulp chambers whole blood and blood with the red cells hemolyzed. The change was manifested in teeth of dogs after freezing, heating, and decomposition in a moist environment. The authors postulate that pink teeth occur as a result of breakdown of red blood cells in the pulp chamber of the tooth and diffusion of hemoglobin and other serum proteins into the dentin via the dential tubules. Histochemical studies show that the brown or gray material in some teeth subjected to postmortem aging is probably hemoglobin and serum proteins. Factors of age, vascularity of the pulp chamber, and postmortem conditions are discussed in relation to the postmortem development of pink teeth.     

Death due to microvascular occlusion in sickle-cell trait following physical exertion

The heterozygous condition characterized by the presence of hemoglobin AS (sickle-cell trait) occurs in approximately 8% of the American black population. Unlike the homozygous state (sickle-cell disease), sickle-cell trait is not widely recognized as a cause of life-threatening illness or death despite over 30 case reports describing fatal or serious complications of exercise in young black males with this condition. These reports identify heat stress, dehydration, viral illness, and poor physical conditioning as factors which may contribute to exertional rhabdomyolysis and sudden death, suggesting multifactorial etiology. However, since sickling is known to occur postmortem, it remains controversial as to whether the pathogenesis of these exercise related deaths involves microvascular obstruction by sickled erythrocytes. We describe three young black individuals with no significant past medical history who died following physical exertion. In all three cases, postmortem hemoglobin electrophoresis demonstrated hemoglobin AS. In none of the cases was the body temperature found to be elevated. These cases serve to remind the forensic community that, in the proper setting, sickle-cell trait must be viewed as a potentially fatal disorder.     

Spectrophotometric evaluation of postmortem lividity

Under low ambient temperatures normally bluish postmortem lividity adopts a bright red or pink colour due to resaturation of haemoglobin with O2. The most important differential diagnosis in the presence of pink hypostasis is carbon monoxide poisoning. To answer the question if objective measuring methods allow differentiation of hypostasis with regard to cold exposition or carbon monoxide poisoning, spectrophotometric measurements were performed and the colorimetric measures as well as the spectral reflectance curves of the postmortem lividity were determined. The colorimetric measures CIE-L*a*b* showed similar values for all bright red livores mortis; differentiation between CO intoxication and cold exposition was not possible. Reflectance curves of pink hypostasis after cold storage showed the typical pattern of O2-rich blood with reflectance minima at wavelengths 541 nm and 576 nm and a reflectance maximum at 560 nm. Pink hypostasis because of carbon monoxide poisoning showed a shift of the reflectance maximum toward 555 nm and a flattened curve in all cases with COHb concentrations exceeding 52%, whereas these changes were not regularly observed with lower COHb levels.     

Biochemical measurements of glucose metabolism in relation to cause of death and postmortem effects

This study was performed to examine the relationship between postmortem biochemical values and cause of death. The follow samples were taken from 399 corpses: cerebrospinal fluid (CSF; n = 376, suboccipital), blood (n = 158, femoral vein), and urine (n = 101, at autopsy). (See Table 1 for causes of death) All samples were stored at -80 degrees C. A further 100 samples of blood were later taken and stored at +4 degrees C before testing. Biochemical determinations made were: glucose in CSF, blood, and urine (hexokinase method); lactate (LDH/GPT) and free acetone (HS-gas chromatography) in CSF; hemoglobin A1 in blood (microcolumn technique). In 34 cases fatal diabetic coma was considered verified by morphological and chemical findings. One hundred cases of sudden cardiac death were chosen as the main control group. In 32 of the 34 cases defined above, the value of the formula of Traub (glucose + lactate in CSF) exceeded 415 mg/dl. It is not influenced significantly by hyperglycemia or hyperlactatemia due to factors other than diabetes (i.e., carbon monoxide, asphyxia). After death the value rose till the 30th hpm, then remained stable for at least 1 week. Fatal coma was defined as the ketoacidotic form if free acetone in CSF ranged above 21 mg/l. In these cases, CSF glucose and free acetone correlated positively. Hemoglobin A1 remained stable after death. Its amount was independent from postmortem blood glucose, postmortem interval and total hemoglobin. Furthermore, the manner of storage (-80 degrees or +4 degrees C) had no significant influence on its values. In 29 of 34 cases of fatal coma, Hb A1 exceeded 12.1%. Analysis of urine glucose showed elevated levels (over 500 mg/dl) in diabetic comas. On conclusion, fatal diabetic coma seems indicated as the cause of death if measured values of postmortem biochemistry exceed the following limits: CSF-Traub 415 mg/dl, free acetone (CSF) 21 mg/l; Hb A1 12.1%; urine glucose 500 mg/dl. Most important are the Traub formula and hemoglobin A1. Usually, in fatal coma both values are elevated. If both of them are normal, diabetic coma can nearly be excluded. Combined evaluation of all values is absolutely necessary. Morphology must also always be taken into account. Consequently, a diagnosis of fatal coma can be obtained by a process of elimination.     

Postmortem formation of carbon monoxide

Since carbon monoxide (CO) production after death was suggested in a drowned body, CO and carboxyhemoglobin (HbCO) levels in blood and body cavity fluids of cadavers which were not exposed to fire and CO have been analyzed. CO released from the tissues was determined by gas chromatography and gas chromatography-mass spectrometry, and the total concentration of hemoglobin (Hb) was measured as cyanmethemoglobin (CNmHb). The HbCO level was calculated by the ratio of CO content and CO-binding capacity. CO levels (ml/100 g at STP) of the seven cases in which blood and body cavity fluids could be collected ranged from 0.13 to 0.87 in blood and 0.02 to 0.80 in body cavity fluids. HbCO levels in blood and body cavity fluids were from 0.3 to 6.0% and from 2.3 to 44.1%, respectively. In a typical case showing postmortem formation of CO, the CO levels in body cavity fluids were higher than that in blood. It is suggested that CO in a putrefied body is due to CO in blood prior to death and the CO formed by the decomposition of Hb, myoglobin and other substances during putrefaction. The significance of HbCO levels in body cavity fluids of cases with marked postmortem decomposition seems difficult to interpret without the value of HbCO in blood.     

Histological and immunohistochemical study of Wischnewsky spots in fatal hypothermia

Wischnewsky spots in the gastric mucosa are considered an important finding for the diagnosis of hypothermia-related deaths. In the present prospective histological and immunohistochemical investigation, 14 cases of fatal hypothermia presenting Wischnewsky spots at autopsy were studied. Macromorphologically, the lesions, varying in diameter from 0.1 to 0.4 cm, had a blackish-brownish color and appeared partly lofty, especially on the apex of gastric folds. Histologically, no erosions or ulcers were observed in the gastric mucosa. In some cases, hemorrhages in conjunction with infarctions of the mucosa were observed in the mucosal glands. Those regions, however, did not represent the lesions visible as Wischnewsky spots at the macroscopical level. Immunohistochemical stains were done with a specific antibody against hemoglobin (Dako, Glostrup, Denmark). Wischnewsky spots expressed immunopositivity with antihemoglobin. Concerning the pathogenesis and underlying pathophysiologic mechanisms contributing to the development of Wischnewsky spots, we hypothesize that cooling of the body in the setting of cold ambient temperatures primarily leads to circumscribed hemorrhages of the gastric glands in vivo or in the agonal period, respectively. Subsequently, due to autolysis, erythrocytes are destroyed and hemoglobin is released. Following exposure to gastric acid, hemoglobin is hematinized, leading to the typical blackish-brownish appearance of Wischnewsky spots seen at gross examination. Wischnewsky spots are not equivalent to erosions in terms of histopathological diagnosis but rather represent epiphenomena generated in vivo or in the agonal period of fatal hypothermia.     

Patho-anatomic findings in neck structures in asphyxiation due to hanging: a survey of 80 cases

By examining 80 consecutive cases of death due to hanging, fractures of the thyroid cartilage or the hyoid bone or both, were noted in 45%. Fracture of the cricoid cartilage did not occur. No fracture occurred below the age of 25, and the frequency was slightly increasing with increasing age. There was a preponderance of fractures in males. The highest incidence of fractures was noted in typical hangings, while incomplete hanging to a very great extent was combined with congestion to the face, especially if the location of the ligature was atypical. Typical hanging in general did not produce congestion. The lividity in the declivous areas became fixed at the earliest after 5-6 h suspension time, while after 12 h almost all the cases demonstrated fixed lividity in the distal parts, especially in the legs. There is slight indication that the frequency of fractures increases with the length of suspension time.     

Time-dependent immunohistochemical detection of proinflammatory cytokines (IL-1β, IL-6, TNF-α) in human skin wounds

The proinflammatory cytokines interleukin-1beta (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) hold important functions in the early and late courses of inflammation, trauma and wound healing. In the present study, human skin wounds due to sharp force (n=105) were collected during surgery and autopsy. The wound age mainly varied from several minutes to 5 h, some specimens aged up to 6 weeks. Control specimens from uninjured skin were available in each case. After preparation of cryostat sections, immunohistochemistry was performed according to the APAAP technique, using monoclonal and polyclonal antibodies. The results were evaluated semiquantitatively. All markers were weakly expressed in normal human skin constitutively. However, the staining pattern changed significantly in vital wounds concerning epidermal layers, subepidermal cells, vessels and sweat glands. IL-1β and IL-6 showed enhanced expression after 15 and 20 min at the earliest (increase of epidermal reactivity). After 30–60 and 60–90 min, respectively, marked expression was observed with these markers. Similar alterations were detectable with TNF-α after 15 and 60–90 min. The reactivity of all three markers persisted over several hours, then decreased to basal levels again and sometimes reappeared after days and in granulation tissue. Leukocytes reacting with IL-1β and IL-6 appeared after approximately 2 h. Conclusion: proinflammatory cytokines can serve as a useful tool for the estimation of vitality and wound age, in particular in the early post-traumatic interval prior to leukocyte reaction. Autolysis did not play a role in the samples investigated (postmortem interval up to 8 days). Problems could sometimes rise from constitutive expression. Therefore, it is recommended to examine control samples from the same individual and to compare the reactivity with wound specimens.     

A preliminary immunohistochemical study of fibronectin in viral myocarditis

For postmortem diagnosis of viral myocarditis, 12 specimens of heart in autopsy were studied immunohistochemically with anti-fibronectin(FN) antibody. Among 5 cases with definite or suspected myocarditis, intensive FN-positive could be found in all monocytes, macrophages and some neutrophils and there were 3 cases with FN-positive cardiomyocytes. While in 3 violent death cases with leucocytes infiltration in the interstitial tissue of myocardia, no FN-positive cardiomocytes were observed, which was the same as that of 4 case without cardiac pathological changes. Moreover, there were rarely FN-positive leucocytes in the 7 cases without viral myocarditis. The results suggest that the immunohistochemical observation of FN in myocardia might be of value for detecting slight degeneration of cardiomyocytes and determining the inflammatory leucocyte infiltration in myocarditis.     

Ethanol formation in unadulterated postmortem tissues

During the investigation of aviation accidents, postmortem samples obtained from fatal accident victims are submitted to the FAA's Civil Aerospace Medical Institute (CAMI) for toxicological analysis. During toxicological evaluations, ethanol analysis is performed on all cases. Many species of bacteria, yeast, and fungi have the ability to produce ethanol and other volatile organic compounds in postmortem specimens. The potential for postmortem ethanol formation complicates the interpretation of ethanol-positive results from accident victims. Therefore, the prevention of ethanol formation at all steps following specimen collection is a priority. Sodium fluoride is the most commonly used preservative for postmortem specimens. Several studies have been published detailing the effectiveness of sodium fluoride for the prevention of ethanol formation in blood and urine specimens; however, our laboratory receives blood or urine in approximately 70% of cases. Thus, we frequently rely on tissue specimens for ethanol analysis. The postmortem tissue specimens received by our laboratory have generally been subjected to severe trauma and may have been exposed to numerous microbial species capable of ethanol production. With this in mind, we designed an experiment utilizing unadulterated tissue specimens obtained from aviation accident victims to determine the effectiveness of sodium fluoride at various storage temperatures for the prevention of microbial ethanol formation. We found that without preservative, specimens stored at 4 degrees C for 96 h showed an increase in ethanol concentration ranging from 22 to 75 mg/hg (average 42 +/- 15 mg/hg). At 25 degrees C, these same specimens showed an increase ranging from 19 to 84 mg/hg (average 45 +/- 22 mg/hg). With the addition of 1.00% sodium fluoride, there was no significant increase in ethanol concentration at either temperature.     

Gross and histologic postmortem changes of the skin

Many methods have been attempted to accurately and systematically determine the estimated time of death in autopsy cases. Gross changes of the skin which occur during the postmortem interval have occasionally been used to estimate the time of death under different conditions; however, the postmortem gross and histologic changes of the skin have not been systematically analyzed. We sequentially studied the gross and microscopic appearance of skin in 3 individuals during the early postmortem interval (<1 week) in an outdoor, shaded, cool to temperate climate. Photos and skin biopsy specimens were taken from 4 sites (acral skin, scalp, dependent truncal skin, and nondependent truncal skin) every 12-24 hours on each individual. The gross appearance of the skin remarkably did not change significantly in any of the sites after 1 week of decomposition. Three main histologic changes were seen in the skin biopsy specimens: focal dermal-epidermal separation, eccrine duct necrosis, and dermal degeneration. Although bullae were not present in the gross examination of the skin, focal separation of the epidermis from the dermis was appreciated in several skin biopsies; however, the timing of development was unpredictable. Eccrine duct necrosis was another histologic finding and was relatively predictable, occurring in dependent and nondependent truncal skin biopsies from all 3 individuals between days 4 and 7. Dermal degeneration was a uniform histologic finding only in biopsies from nondependent truncal skin, occurring in all individuals on day 2. Although this study is limited by the small number of subjects and uncontrolled extrinsic factors, histologic findings, such as eccrine gland necrosis and dermal degeneration, in select cutaneous biopsies may be useful in estimating the time of death in the early postmortem interval.