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Background

Post-mortem biochemistry, including the analysis of beta-hydroxybutyrate (BHB), is increasingly employed in forensic medicine, especially in conditions such as diabetes and chronic alcoholism. However, not much is known about the associations between age, body mass index (BMI), and sex and BHB concentrations in ketoacidotic conditions.

Aim

To retrospectively study the association between age, BMI and sex in several conditions, such as diabetic ketoacidosis (DKA), alcoholic ketoacidosis (AKA), and elevated post-mortem BHB concentrations.

Methods

1407 forensic autopsy cases analysed for BHB were grouped by diagnosis: DKA, AKA, HHS [hyperosmolar hyperglycaemic state], acidosis NOS [not otherwise specified], or hypothermia. Age, sex, BMI and the concentrations of blood alcohol, vitreous glucose and blood BHB were recorded.

Results

Cases of AKA and DKA were most numerous (184 and 156, respectively). In DKA and in its male subgroup, cases with severe ketosis (BHB>1000 µg/g) were younger and had a lower BMI than those with moderate ketosis (BHB 250–1000 µg/g) and controls (P<0.001). In DKA and in its female subgroup, cases with moderate ketosis cases were older (P = 0.0218 and P = 0.0083) than controls. In AKA and in its male subgroup, cases with severe ketosis had a lower BMI than those with moderate ketosis (P = 0.0391 and P = 0.0469) and controls (P<0.001). Cases with moderate ketosis had a lower BMI than controls (P<0.001).

Conclusions

BHB concentration is associated with BMI in DKA and AKA, and with both BMI and age in DKA. Constitutional factors should, therefore, be considered in potential AKA and DKA cases.

Background. Hyaluronan (HA) is a component of the extracellular matrix in lung tissue and is normally present at low concentrations in blood. HA is rapidly cleared from blood by the liver. Increased concentrations of plasma HA have been found in patients with acute respiratory distress syndrome (ARDS). We investigated changes in HA levels in plasma, bronchoalveolar lavage fluid (BALF), and lung, and their relationship to pretreatment with a leukocyte elastase inhibitor in a rat model of ARDS.

Methods. Rats were randomly assigned to three groups: control, thrombin, and thrombin plus elastase inhibitor. By use of a radiometric assay, HA was measured in lungs, BALF, and plasma. Tissue samples from the lungs were stained for HA and examined microscopically. Liver circulation and cardiac output were monitored using radiolabeled microspheres.

Results. Infusion of thrombin produced a pronounced increase in wet weight to dry weight ratio, and relative lung water content. This increase was blunted by a leukocyte elastase inhibitor. A decrease in lung HA and increases in both BALF and plasma HA were found. The leukocyte elastase inhibitor counteracted not only the decrease in lung tissue HA, but also the increase in plasma HA. Histologically, there was decreased HA-staining of peribronchial and perivascular areas in the injured rat lung. Decreased liver perfusion was observed after infusion of thrombin.

Conclusions. The decrease in lung HA may be involved in the development of pulmonary edema in this ARDS model, and leukocyte elastase may be one cause of this decrease. In addition, an elevated plasma HA level may be an indicator of lung injury.

The preventive effect of indomethacin on thrombin-induced pulmonary edema was studied in rats. Administration of thrombin caused a significant increase in lung weight, wet weight to dry weight ratio (WW/DW), and relative lung water content. During infusion of thrombin, mean pulmonary artery pressure rose and mean systemic artery pressure fell, PaO₂ decreased progressively and there was a continuous rise in pH and PaCO₂.

An inhibitor of cyclooxygenase, indomethacin, at a dose of 1 mg/kg body weight, induced a significant further increase in lung weight (p<0.05), and a tendency towards an increase in WW/DW and water content compared with animals given thrombin alone. Treatment with indomethacin, however, counteracted the elevated pulmonary artery pressure occurring in the early phase after thrombin infusion, but not that in the late phase. Systemic artery pressure was not affected by indomethacin. The increases in pH and PaCO₂ after thrombin infusion were attenuated and remained stable almost at baseline level after indomethacin administration. Indomethacin did not prevent the hypoxemia induced by thrombin infusion.

In conclusion, although indomethacin prevented the early increase in pulmonary artery pressure due to thrombin and the decrease in pH and the increase in PaCO₂, it caused lung vascular permeability to protein to increase more than with thrombin alone.

The effect of a selective leukocyte elastase inhibitor, ICI 200,355, on thrombin-induced pulmonary oedema was studied in rats. Thrombin administration produced an increase in lung weight (P < 0.05), wet weight/ dry weight ratio (P < 0.05), and relative lung water content (P < 0.05). The lung weight increase was reduced by the elastase inhibitor in doses of 2000, 200 and 20 micrograms/kg per h (P < 0.05), but not by 2 micrograms/kg per h. A dose of 20 micrograms/ kg per h seems to be optimal, since 10-fold and 100-fold increases in dose did not further improve the effect. Free elastase activity in lung tissue was higher after thrombin infusion than in controls, but was not depleted by the elastase inhibitor in vivo (P < 0.05). This elastase activity in the lung was, however, inhibited by the elastase inhibitor in vitro, indicating that the inhibitor can block extracellular, but not intracellular elastase activity. Thrombin infusion resulted in a significant decrease in plasma elastase inhibitory capacity (P < 0.05), which was depleted by the elastase inhibitor (20 micrograms/kg per h) (P < 0.05). Myeloperoxidase activity was significantly increased in lung tissue after thrombin infusion (P < 0.05). Lung myeloperoxidase activity 5 min after thrombin infusion was not affected by the elastase inhibitor, but the inhibitor induced a further increase in myeloperoxidase as seen 90 min after thrombin infusion, indicating that the effect of this inhibitor on pulmonary oedema is not due to reduction of leukocyte infiltration in the lungs, but may partly be exerted by prevention of neutrophil destruction.

The effect of ibuprofen on thrombin-induced pulmonary edema was studied in rats. Thrombin infusion produced a significant increase in lung weight, wet weight/dry weight ratio and relative lung water content, a rise in mean pulmonary arterial pressure and a fall in mean systemic arterial pressure. It also caused a progressive decrease in PaO2 and a continuous increase in pH and PaCO2. Administration of either the S-isomer or R-isomer of ibuprofen at doses of 5 mg/kg body weight prior to thrombin infusion resulted in significant reduction in lung weight, wet weight/dry weight ratio and water content. The wet weight/dry weight ratio and the water content were somewhat lower after infusion of the S-isomer than of the R-isomer. Ibuprofen diminished the thrombin-induced increase in mean pulmonary arterial pressure and attenuated the early and late decrease in mean systemic arterial pressure caused by thrombin. Ibuprofen also stabilized thrombin-induced impairments in PaO2, PaCO2 and pH. The results thus indicate that ibuprofen effectively counteracts hemodynamic changes, stabilizes impairments in arterial blood gas variables and attenuates the increase in lung vascular permeability to protein with pulmonary edema caused by thrombin. The results also indicate a substantial R to S chiral inversion of ibuprofen in vivo in the rat.

Sequencing of mitochondrial DNA (mtDNA) has been used for human identification based on teeth and skeletal remains. Here, we describe an amplification system for the mtDNA control region (D-loop) suited for the analysis of shed hair, which constitutes the most common biological evidence material in forensic investigations. The success rate was over 90% when applied to evidence materials such as shed hair, saliva stains and saliva on stamps. The analysis of evidence materials collected from three similar robberies revealed the presence of mtDNA sequences identical to those of the suspects in the three crimes. The use of mtDNA control region sequences for individual identification was evaluated. The probability of identity by chance for the mtDNA types of the suspects in the robberies was found to vary between Pr = 0.017 - < 0.0017, depending on the reference population used, emphasizing the need for large population databases to obtain the appropriate estimate.

A major problem in analyzing forensic casework samples is the presence of genetic material from more than one individual in the material evidence. For instance, in sexual assault cases the evidence (vaginal swabs) usually contains a majority of vaginal epithelial cells and varying amounts of sperm cells from the perpetrator. Samples with mixed genotypes are also common among other biological evidence materials such as nail scrapes and mixed bloodstains. We have developed an allele-specific amplification system for the highly polymorphic HLA class II DRB1 locus that permits the detection of individual alleles in a sample with mixed genotypes, independent of the initial frequency of the alleles. Using a set of eight allele-specific amplification primers and typing the amplified fragments with sequence-specific probes, most of the 60 DRB1 alleles can be resolved. The method is highly specific and sensitive, with the potential for amplifying 15 copies of a particular allele in a background of 3 x 10(5) copies of other alleles. The method was successfully applied to three forensic cases, where the material evidence consisted of sperm stains on panties, nail scrapes and bloodstains on skin. Thus the DRB1 allele-specific amplification system can be employed for the unambiguous determination of the presence of individual alleles in materials suspected to contain mixed genotypes, even when the alleles of interest constitute only a small fraction of the total DNA

In forensic cases involving mail bombs, extortion, kidnapping or threatening letters, biological evidence such as the saliva used to attach the stamp and seal the envelope could be used for genetic analysis. We have developed a highly sensitive semi-nested PCR method for the HLA-DRB1 locus; suitable for the analyses of very limited amounts of DNA. When applied to a set of stamps and envelopes with saliva from control individuals, typing results were consistent with those obtained using hairs drawn from the same individuals. No interference was found due to DNA from the fingerprints of people handling the letters. The system was applied to three forensic cases with threatening letters. The first case resulted in an exclusion of the suspect. In the second case, the suspect could not be excluded (probability of identical genotype by chance > 0.01). These results demonstrate that biological evidence in cases with threatening letters is amenable to genetic typing.

According to scientific studies, subdural haematoma (SDH) in combination with retinal haemorrhage and encephalopathy (usually denoted as the triad) during infancy is highly specific for abusive head trauma/shaken baby syndrome, if a fall from over one meter, a traffic accident and certain medical conditions have been excluded. Other studies have challenged this notion since there are witnessed falls from less than one meter which has caused acute SDH, birth-related SDH which have been confirmed to develop into chronic SDH, and external hydrocephalus that can be complicated by a spontaneous SDH or an SDH from minor trauma.

These aetiologies have not been taken into consideration in prior studies on abusive head trauma. 

An independent review on infant abusive shaking from the Swedish Agency for Health Technology Assessment concluded that there was insufficient evidence for determining the specificity of the triad for isolated shaking. 

The aim of this thesis was to increase the understanding of the aetiologies and the epidemiology of infant SDH. This was accomplished by studying registry data (Paper I and II), descriptive reviews of infants with SDH (paper I, III and IV) and infants subjected to witnessed or spontaneously admitted shaking (paper V).

The maximum incidence of fatal AHT in Sweden, 0.6/100 000, was at least 10 times lower than in other Western countries and the risk of unreported fatal AHT was low (Paper I). A majority of the deceased infants had neonatal conditions, several were twins and/or preterm, one had a cerebral vascular condition, and one signs of metabolic disorder (Paper I). Approximately 12 infants every year are diagnosed with SDH beyond the first week of life and the case-fatality rate for all SDH diagnosis categories is 6.2% (Paper II). Males, preterm born and twins have a higher risk to develop SDH in general, and those having an abuse diagnosis had increased odds of being born preterm and small-for-gestational age (Paper II). Chronic SDH has a higher freqency of male and premature infants and a lower mortality rate than acute SDH (Paper III). Infants with chronic CSDH, but not with acute SDH, tend to have findings on neuroimaging and a head circumference that are suggestive of external hydrocephalus complicated by spontaneous SDH or SDH from minor trauma (Paper IV). Intracranial and ocular findings in infants subjected to abusive shaking were rare, seen in 2 out of 36 infants, both with pre-existing intracranial pathology, and of non-specific character (Paper V).

In conclusion this thesis provides evidence that non-abusive aetiologies for SDH in infancy may have been overlooked in previous research.   

AIM: The validity of the diagnostic criteria for abusive head trauma (AHT) and its attributes have been widely debated. This national study investigated the possibility of false positive and false negative cases of fatal AHT in Sweden.

METHOD: This was a retrospective evaluation of the records of 733 deceased infants up to the age of 365 days who were examined during 1994-2013 at the six forensic medicine departments. All the records were scrutinised for possible cases of AHT.

RESULTS: We included 12 cases, out of which eight had been diagnosed as AHT. Of these 12 infants, eight had a concomitant disease or perinatal illness, five were born prematurely, and three were twins. Figures from other Western countries would suggest 6-7 deaths per 100,000 per year in Sweden but in reality, there was a maximum possible incidence of 0.6 per 100,000 infants per year.

CONCLUSION: The risk of unreported fatal AHT in Sweden was low and there may have been cases misdiagnosed as AHT. The at least 10 times lower incidence than has been reported from other Western countries, raises the question if previously reported higher incidences of fatal AHT have been exaggerated.

Background Acute (ASDH) and chronic subdural haematoma (CSDH) in infants have been regarded as highly specific for abuse. A recent study showed different risk factors for ASDH and CSDH, indicating that CSDH in many cases was related to external hydrocephalus. 

Purpose To investigate to what extent external hydrocephalus may explain findings and symptoms interpreted as signs of abusive head trauma. 

Material and methods Eighty-five infants with ASDH (n=16) and CSDH (n=69) were reviewed with regard to cranio-cortical- (CCW), sino-cortical- (SCW), frontal interhemispheric-(IHW), subarachnoid space width (SSW) and head circumference (HC). In infants with unilateral SDH, the correlation between the contralateral SSW and the ipsilateral CCW and SDH width was calculated. A correlation would imply that the CSDH replaces an already existing extracerebral space.

Results Infants with CSDH had significantly higher CCW, SCW, IHW and SSW than infants with ASDH (p < 0.05). The ipsilateral CCW (R = 0.92, p < 0.001) and SDH width (R = 0.81, p < 0.01) were correlated to the contralateral SSW. Increased HC was more prevalent in Infants with CSDH (71%) than in infants with ASDH (14%) (p < 0.01). Forty-two infants, all with CSDH, had at least one of CCW, SCW or IHW ≥ 95^th percentile. Twenty infants, all with CSDH, had CCW, SCW and IHW > 5 mm and increased HC. 

Conclusion A significant proportion of infants with CSDH may have external hydrocephalus as an underlying cause and that parts of the widened subarachnoid space in some infants is replaced by a CSDH.

Background: In a register study based on ICD 10 coding, there was a similar vulnerability profile (male sex, preterm and small for gestational age) in infants diagnosed with non-traumatic subdural haematoma (SDH) and infants having SDH with abuse diagnosis. However, ICD-10 does not separate between acute (ASDH) and chronic subdural haematoma (CSDH). 

Purpose: To determine the vulnerability profile in infants having CSDH and ASDH, respectively. 

Material and methods: A descriptive review of infants with SDH/hygroma examined by the Swedish National Board of Forensic Medicine between 1994 and 2018. Included cases (n=85) were analysed with regard to possible vulnerability factors. 

Results: Type of subdural fluid could be determined in 85 of 96 cases. Sixteen infants had ASDH and 69 CSDH. Infants with ASDH had the peak incidence during the first month of life, 56% were male, 6% were premature, 13% were twins and 44% died. In infants with CSDH, the peak incidence occurred during the third month of life, 69% were male, 34% were premature, 12% were twins and 4% died. 

Conclusion: CSDH, but not ASDH, is associated with factors suggesting non-traumatic pathogenesis, for which reason CSDH and ASDH should be analysed separately to extend the knowledge regarding the aetiology of SDH during infancy.  

BACKGROUND: Acute subdural hematoma (ASDH) and chronic subdural hematoma (CSDH) in infants have been regarded as highly specific for abuse. Other causes of CSDH have not been investigated in a large population.

PURPOSE: The purpose of this study was to investigate to what extent external hydrocephalus is present in infants with ASDH and CSDH undergoing evaluation for abuse.

MATERIAL AND METHODS: Eighty-five infants suspected of being abused, with ASDH (n = 16) or CSDH (n = 69), were reviewed regarding age, risk factor profiles, craniocortical width (CCW), sinocortical width (SCW), frontal interhemispheric width (IHW), subarachnoid space width (SSW), and head circumference (HC). In infants with unilateral subdural hematoma (SDH), correlations between contralateral SSW and ipsilateral CCW and SDH width were investigated.