Autopsy assessment of pediatric head injury: a proposal for aerosol mitigation during the COVID-19 pandemic.

During the Corona Virus Disease-19 (COVID-19) pandemic, there is still a requirement for post-mortems to continue, including those examinations performed in the context of medico-legal investigations. Currently, very little is known about how long this coronavirus can survive in deceased human bodies or whether un-embalmed human cadavers can be contagious to people who handle them. Therefore, it would appear to be prudent to consider implementation of additional safety measures for all necessary post-mortem procedures. During the post-mortem examination of babies and young children, it is important to open the calvarium to enable visualization of the brain and its coverings, particularly in cases where a head injury is likely to have occurred. Since October 2013, the use of neurosurgical equipment to open the calvarium during infant and young child autopsies has become routine practice in our unit. Both the neurosurgical craniotome and a standard oscillating mortuary saw produce particulate matter consisting of bone and body fluids (including blood) which can become aerosolized. Within this paper, we discuss the use of a transparent plastic tent whilst opening the calvarium during pediatric post-mortems, to reduce the spread of aerosols into the mortuary environment.

Frequency of macroscopic intradural hemorrhage with and without subdural hemorrhage in early childhood autopsies.

Some authors have suggested that in the fetus, neonate and infant, intradural hemorrhage (IDH) is relatively common and often presents alongside subdural hemorrhage (SDH). These authors have theorized that pediatric SDH may result from an IDH due to blood leakage from a dural vascular plexus. In this study, we report the inter-observer variation for detection of IDH from a retrospectively collected series of pediatric autopsy photographs, with and without SDH. Autopsy photographs of the falx and tentorium from 27 neonatal, infant and early childhood autopsies were assessed by two independent consultant forensic pathologists blinded to all case histories for the presence and extent (focal or diffuse) of IDH. Inter-observer agreement between the pathologists was calculated using Cohen's kappa coefficient. The occurrence of subdural hemorrhage was also recorded at autopsy. A kappa coefficient value of 0.669 (p = 0.001), indicated a substantial level of agreement for the presence/absence of IDH between the pathologists. For the extent of IDH a kappa coefficient value of 0.6 (p = 0.038) indicated a moderate level of agreement. The pathologists agreed on the presence of IDH in 10 of the 27 cases. Subdural hemorrhage was recorded for 8 out of 27 cases. Of these 8 cases, it was agreed that 4 had IDH. Using standardized methods of image capture and assessment, inter-observer agreement for the presence/absence of IDH was substantial. In this paper, we report a much lower frequency of macroscopic IDH occurring alongside SDH than previous studies, which included both gross observation of IDH and histological examination.

A systematic autopsy survey of human infant bridging veins.

In the first years of life, subdural haemorrhage (SDH) within the cranial cavity can occur through accidental and non-accidental mechanisms as well as from birth-related injury. This type of bleeding is the most common finding in victims of abusive head trauma (AHT). Historically, the most frequent cause of SDHs in infancy is suggested to be traumatic damage to bridging veins traversing from the brain to the dural membrane. However, several alternative hypotheses have been suggested for the cause and origin of subdural bleeding. It has also been suggested by some that bridging veins are too large to rupture through the forces associated with AHT. To date, there have been no systematic anatomical studies on infant bridging veins. During 43 neonatal, infant and young child post-mortem examinations, we have mapped the locations and numbers of bridging veins onto a 3D model of the surface of a representative infant brain. We have also recorded the in situ diameter of 79 bridging veins from two neonatal, one infant and two young children at post-mortem examination. Large numbers of veins, both distant from and directly entering the dural venous sinuses, were discovered travelling between the brain and dural membrane, with the mean number of veins per brain being 54.1 and the largest number recorded as 94. The mean diameter of the bridging veins was 0.93 mm, with measurements ranging from 0.05 to 3.07 mm. These data demonstrate that some veins are extremely small and subjectively, and they appear to be delicate. Characterisation of infant bridging veins will contribute to the current understanding of potential vascular sources of subdural bleeding and could also be used to further develop computational models of infant head injury.

Post-mortem imaging of the infant and perinatal dura mater and superior sagittal sinus using optical coherence tomography.

Infants and young children are likely to present with subdural haemorrhage (SDH) if they are the victims of abusive head trauma. In these cases, the most accepted theory for the source of bleeding is the bridging veins traversing from the surface of the brain to the dura mater. However, some have suggested that SDH may result from leakage of blood from a dural vascular plexus. As post-mortem examination of the bridging veins and dura is challenging, and imaging modalities such as magnetic resonance and computed tomography do not have the resolution capabilities to image small blood vessels, we have trialled the use of intravascular and benchtop optical coherence tomography (OCT) systems for imaging from within the superior sagittal sinus (SSS) and through the dura during five infant/perinatal autopsies. Numerous vessel-like structures were identified using both OCT systems. Measurements taken with the intravascular rotational system indicate that the approximate median diameters of blood vessels entering anterior and posterior segments of the SSS were 110 µm (range 70 to 670 µm, n = 21) and 125 µm (range 70 to 740 µm, n = 23), respectively. For blood vessels close to the wall of the SSS, the median diameters for anterior and posterior segments of the SSS were 80 µm (range 40 to 170 µm, n = 25) and 90 µm (range 30 to 150 µm), respectively. Detailed characterisation of the dural vasculature is important to aid understanding of the source of SDH. High resolution 3-dimensional reconstructions of the infant dural vasculature may be possible with further development of OCT systems.

Visualisation of the intact dura mater and brain surface in infant autopsies: a minimally destructive technique for the post-mortem assessment of head injury.

During the post-mortem examination of babies and young children, it is important to be able to visualise the brain and its coverings, particularly in cases where a head injury is likely to have occurred. In this paper, we present an improved method for removal of the calvarial bones in infant autopsies to enable viewing of the dura mater and brain. In contrast to the standard post-mortem procedure for observing and removing the brain, this novel technique is minimally disruptive, allowing the dura mater to remain undamaged. Specialised paediatric neurosurgical tools were used to remove the skull bones from 23 neonates, infants and young children during post-mortem examination. In 21 of our 23 cases, the calvarial bones were removed successfully with the dura mater remaining intact. In one case, there was a thickening of the dura mater which created a strong adherence of this membrane to the bone. In another case, the dura mater was slightly damaged due to the inexperience of the operator in using the neurosurgical tools. This method of calvarial bone removal reduces the degree of post-mortem artefact and enhances the ability to observe and photographically document autopsy findings, including the artefact-free detection of signs of injury such as epidural or subdural haematoma, and brain swelling. This technique has now become a routine practise in both of our units to remove the skull bones in infant/young children post-mortem examinations.

Optical clearing of the dura mater using glycerol: a reversible process to aid the post-mortem investigation of infant head injury.

PURPOSE: In cases of suspected abusive head trauma, a thorough and systematic study of the cranium and its contents is essential, preferably using the best available methods for observing the brain and its coverings. Building upon recent developments in skull bone removal techniques in infant autopsies, we have assessed the use of two optical clearing agents (OCAs), glycerol and mannitol, on pediatric dura mater in an attempt to increase the transparency of this tissue and thereby enhance the post-mortem assessment of infant head injuries, particularly subdural hematomas. METHODS: Extracorporeal testing revealed glycerol to be the more effective OCA. Therefore, in situ investigations were commenced using glycerol during 33 pediatric post-mortem examinations. RESULTS: An increase in the transparency of the dura was observed in 32 of the 33 cases, within 1 min of application of the OCA. In a 2 year old with cerebral palsy, only partial optical clearance of the dura was seen, most likely due to a significantly atrophic brain, prominent gelatinous leptomeninges, and abnormally thickened dura. This technique allowed for detection of minimal amounts of subdural bleeding over the convexities, before dissection of the dura, avoiding post-mortem blood spillage from artifactually disrupted bridging veins. Optical clearing of the dura aided in the evaluation of patterns of subdural hemorrhage in three cases of non-accidental head injury, three cases of peri-natal head injury and one case of overlaying, apparently resulting in minor crush injury to the head. CONCLUSIONS: We have demonstrated that glycerol is an effective and easy-to-use OCA to effect the readily reversible optical clearing of human infant calvarial dura at autopsy.

Macroscopic and histological examination of human bridging veins.

In infantile abusive head injury (AHT), subdural haemorrhage (SDH) is commonly held to result from traumatic damage to bridging veins traversing from the surface of the brain to the dura and dural venous sinuses. However, there are limited published radiological or autopsy demonstrations of ruptured bridging veins and several authors also assert that bridging veins are too large to rupture due to the forces associated with AHT. There have been several studies on the size, locations and numbers of adult bridging veins and there is one small study of infant bridging veins. However, there are no microscopic studies of infant bridging veins and only a select few ultrastructural investigations of adult bridging veins. Hitherto, it has been assumed that bridging veins from infants and younger children will display the same anatomical characteristics as those in adulthood. At 19 neonatal, infant and young child post-mortem examinations, we macroscopically examined and sampled bridging veins for microscopy. We compared the histology of those samples with bridging veins from an older child and two adults. We demonstrate that adult bridging veins are usually surrounded by supportive meningeal tissue that appears to be lacking or minimally present around the bridging veins of younger children. Neonatal, infant and young children's veins had a free 'bridging' section. Neonatal and infant bridging veins had smaller diameter ranges and thinner walls (some only 5-7 µm) than those seen in older children and adults. Bridging vein walls contained both fine strands of elastic fibers and a more pronounced elastic lamina. The presence of an elastic lamina occurred more frequently in the older age groups These anatomical differences between the veins of adults and young children may help to explain apparent increased vulnerability of neonatal/infant bridging veins to the forces associated with a shaking-type traumatic event.

Lipid biomarkers of glioma cell growth arrest and cell death detected by 1 H magic angle spinning MRS.

Biomarkers of early response to treatment have the potential to improve cancer therapy by allowing treatment to be tailored to the individual. Alterations in lipids detected by in vivo MRS have been suggested as noninvasive biomarkers of cell stress and early indicators of cell death. An improved understanding of the relationship between MRS lipids and cell stress in vitro would aid in the translation of this technique into clinical use. Rat BT4C glioma cells were treated with 50 µ m cis-dichlorodiammineplatinum II (cisplatin), a commonly used chemotherapeutic agent, and harvested at several time points up to 72 h. High-resolution magic angle spinning (1) H MRS of cells was then performed on a 600-MHz NMR spectrometer. The metabolites were quantified using a time domain fitting method, TARQUIN. Increases were detected in saturated and polyunsaturated fatty acid resonances early during the exposure to cisplatin. The fatty acid CH(2) /CH(3) ratio was unaltered by treatment after allowing for contributions of macromolecules. Polyunsaturated fatty acids increased on treatment, with the group -CH=CH-CH(2) -CH=CH- accounting for all the unsaturated fatty acid signals. Transmission electron microscopy, in addition to Nile red and 4',6-diamino-2-phenylindole co-staining, revealed that the lipid increase was associated with cytoplasmic neutral lipid droplets. Small numbers of apoptotic and necrotic cells were detected by trypan blue, annexin V-fluorescein isothiocyanate-labelled flow cytometry and DNA laddering after up to 48 h of cisplatin exposure. Propidium iodide flow cytometry revealed that cells accumulated in the G1 stage of the cell growth cycle. In conclusion, an increase in the size of the lipid droplets is detected in morphologically viable cells during cisplatin exposure. (1) H MRS can detect lipid alterations during cell cycle arrest and progression of cell death, and has the potential to provide a noninvasive biomarker of treatment efficacy in vivo.

C3 Glomerulopathy and Related Disorders in Children: Etiology-Phenotype Correlation and Outcomes.

BACKGROUND AND OBJECTIVES: Membranoproliferative GN and C3 glomerulopathy are rare and overlapping disorders associated with dysregulation of the alternative complement pathway. Specific etiologic data for pediatric membranoproliferative GN/C3 glomerulopathy are lacking, and outcome data are based on retrospective studies without etiologic data. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A total of 80 prevalent pediatric patients with membranoproliferative GN/C3 glomerulopathy underwent detailed phenotyping and long-term follow-up within the National Registry of Rare Kidney Diseases (RaDaR). Risk factors for kidney survival were determined using a Cox proportional hazards model. Kidney and transplant graft survival was determined using the Kaplan-Meier method. RESULTS: Central histology review determined 39 patients with C3 glomerulopathy, 31 with immune-complex membranoproliferative GN, and ten with immune-complex GN. Patients were aged 2-15 (median, 9; interquartile range, 7-11) years. Median complement C3 and C4 levels were 0.31 g/L and 0.14 g/L, respectively; acquired (anticomplement autoantibodies) or genetic alternative pathway abnormalities were detected in 46% and 9% of patients, respectively, across all groups, including those with immune-complex GN. Median follow-up was 5.18 (interquartile range, 2.13-8.08) years. Eleven patients (14%) progressed to kidney failure, with nine transplants performed in eight patients, two of which failed due to recurrent disease. Presence of >50% crescents on the initial biopsy specimen was the sole variable associated with kidney failure in multivariable analysis (hazard ratio, 6.2; 95% confidence interval, 1.05 to 36.6; P<0.05). Three distinct C3 glomerulopathy prognostic groups were identified according to presenting eGFR and >50% crescents on the initial biopsy specimen. CONCLUSIONS: Crescentic disease was a key risk factor associated with kidney failure in a national cohort of pediatric patients with membranoproliferative GN/C3 glomerulopathy and immune-complex GN. Presenting eGFR and crescentic disease help define prognostic groups in pediatric C3 glomerulopathy. Acquired abnormalities of the alternative pathway were commonly identified but not a risk factor for kidney failure.

Degos disease: a new simulator of non-accidental injury.

Recent high-profile cases have made paediatricians very aware of the serious implications of either missing or wrongly diagnosing non-accidental injury. Subdural fluid collections in non-mobile infants usually represent haemorrhage caused by non-accidental injury. We report a 6-month-old male who presented to the Accident and Emergency Department of Birmingham Heartlands Hospital with bilateral subdural fluid collections and skin ulcers resembling cigarette burns. Non-accidental injury was considered to be the most likely diagnosis. However, while under observation in hospital, the child's neurological condition deteriorated with progressive cerebral infarctions, and serial photographs of the skin lesions showed failure to heal. The revised diagnosis, confirmed histologically, was Degos disease, an extremely rare and often fatal occlusive vasculopathy. The child was treated palliatively and died 8 weeks after presentation. This report informs doctors of a new simulator of non-accidental injury to be considered in infants with otherwise unexplained subdural fluid collections.

Detection of bacterioplankton using PCR probes as a diagnostic indicator for drowning; the Leicester experience.

Bodies found immersed in water can pose difficulties to the investigating authorities. Pathologists may be assisted with the diagnosis by the use of tests such as the analysis for diatoms or the levels of strontium in the blood, although there is a recognised level of uncertainty associated with these tests. Recent work from Japan has shown that using molecular approaches, most recently real-time polymerase chain reaction (PCR) assays with TaqMan probes for bacterioplankton, it is possible to undertake rapid, less laborious, high throughput tests to differentiate freshwater from marine bacterioplankton and in doing so provide a molecular diagnostic test to assist in the diagnosis of drowning. We report the experiences of a United Kingdom forensic pathology unit in the use of this PCR based system for the diagnosis of drowning. We applied this technique to 20 adult and child cadavers from 4 bath, 12 freshwater, 2 brackish and 2 salt water scenes both from within the United Kingdom and abroad. Drowning was concluded to be the cause of death in 16 of these 20 cases and the PCR method supported this conclusion in 12 of these 16 cases. The PCR did not provide evidence of drowning in the four cases where death was from other causes. We illustrate that this PCR method provides a rapid diagnostic supportive test for the diagnosis of drowning that can be applied to United Kingdom autopsy practice.

Molecular screening for colon cancer in inflammatory bowel disease.

Prophylactic colectomy is an established method of bowel cancer prevention in high-risk patients such as familial adenomatosis polyposis coli (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) kindreds, as well as in patients with longstanding ulcerative colitis or Crohn's disease. The decision to perform such a major procedure cannot be made lightly, and issues of timing are important. In inflammatory bowel disease (IBD), the trigger to carry out colectomy usually relates to detection of dysplasia, well recognized to be a premalignant lesion. Screening methods are aimed at detection of precursor lesions or clinically occult malignancies. In current clinical practice, such screening methods are either resource intensive (e.g. colonoscopy and biopsy) or of low sensitivity and specificity (e.g. faecal occult blood testing). We discuss the development of screening techniques based upon molecular technologies and the limitations engendered by our incomplete understanding of the natural history and molecular biology of colorectal neoplasia in IBD.

1H magnetic resonance spectroscopy metabolites as biomarkers for cell cycle arrest and cell death in rat glioma cells.

BACKGROUND: Improved non-invasive imaging biomarkers of treatment response contribute to optimising cancer management and metabolites detected by proton magnetic resonance spectroscopy ((1)H MRS) show promise in this area. Understanding (1)H MRS changes occurring in cells during cell stress and cell death in vitro should aid the selection of pertinent biomarkers for clinical use. METHODS: BT4C glioma cells in culture were exposed to either 50 µM cis-dichlorodiammineplatinum II (cisplatin) or starvation by culture in phosphate buffered saline. High resolution magic angle spinning (1)H MRS was performed on cells using a Varian 600 MHz nanoprobe and metabolites were quantified by a time domain fitting method. Cell viability was assessed by trypan blue, H&E, 4',6-diamino-2-phenylindole (DAPI), DNA laddering and annexin V-FITC labelled flow cytometry; propidium iodide flow cytometry was used to assess the cell cycle phase. RESULTS: With cisplatin exposure, cells initially accumulated in the G1 stage of the cell cycle with low numbers of apoptotic and necrotic cells and this was associated with decreases in phosphocholine, succinate, alanine, taurine, glycine and glutamate and increases in lactate and glycerophosphocholine (GPC). Starvation, leading to necrotic cell death within 6-18 h, caused decreases in succinate, alanine, glycine, and glutamate and increases in GPC. Principal component analysis revealed two patterns of metabolite changes, one common to both types of cell stress and another specific for necrosis secondary to cell starvation. CONCLUSIONS: (1)H MRS reveals alterations in multiple metabolites during cell cycle arrest and cell death which may provide early biomarker profiles of treatment efficacy in vivo.

ERbeta isoform expression in colorectal carcinoma: an in vivo and in vitro study of clinicopathological and molecular correlates.

Colorectal carcinoma shows several sex-related differences with regard to incidence, response to chemotherapy and microsatellite instability. These differences may relate to differential expression of ERbeta1 (wild-type) as well as the truncated ERbeta2 and ERbeta5 splice variant isoforms, which have recently been detected in normal and malignant colorectal epithelium. This hypothesis was tested through the study of ERbeta isoform protein and/or mRNA expression amongst 91 primary colorectal carcinoma cases and 20 colorectal carcinoma cell lines. Study of the latter showed an absolute correlation between mRNA and protein expressions for ERbeta1 and ERbeta2. ERbeta1 and ERbeta2 protein expression was lost in 22% and 49%, respectively, of the primary colorectal carcinomas. By contrast, ERbeta5 expression was found in all primary colorectal carcinomas and all colorectal carcinoma cell lines studied. Lower ERbeta1 protein expression was associated with poorer differentiation, higher pT stage and absence of microsatellite instability. Higher ERbeta2 protein expression was associated with right-sided location and presence of lymph node metastases. Protein expression of ERbeta1 correlated positively with expression of the oestrogen-responsive protein trefoil factor 1 (TFF1). There was no correlation between ERbeta protein isoform expression and response to 5-fluorouracil therapy, tumour proliferation, or thymidylate synthase expression. These data suggest that ERbeta1 and/or ERbeta2 isoform expression may have prognostic value and may explain sex-related differences in microsatellite instability and colorectal carcinoma. The opposing associations shown by ERbeta1 and/or ERbeta2 in relation to colorectal carcinoma are in keeping with differential activities shown by the two isoforms.

Expression of Sonic hedgehog pathway genes is altered in colonic neoplasia.

The Hedgehog (Hh) signalling pathway is crucial for normal development and patterning of numerous human organs including the gut. Hh proteins are also expressed during gastric gland development and gastric epithelial differentiation in adults. Recently, dysregulation of these developmentally important genes has been implicated in cancer, leading to the present study of the expression of Hh signalling proteins in colon cancer. In this study, normal colon and colonic lesions (hyperplastic polyp, adenoma, and colonic adenocarcinoma) were examined by immunohistochemistry using antibodies against Hh signalling molecules: the secreted protein Sonic hedgehog (SHH), its receptor Patched (PTCH), and the PTCH-associated transmembrane protein Smoothened (SMOH). The study shows that Hh signalling pathway members are expressed in normal colonic epithelium. SHH was expressed at the top of the crypts and in a few basally located cells, while PTCH was detected in the neuroendocrine cells and SMOH at the brush border of superficial epithelium. RT-PCR analysis of laser-microdissected crypts from normal human colon confirmed that mRNAs encoding these proteins were expressed in colonic epithelium. Expression of SHH, PTCH, and SMOH was up-regulated in hyperplastic polyps, adenomas, and adenocarcinomas of the colon, and SHH expression correlated with increased expression of the proliferation marker Ki-67 in all lesions examined. To address whether the Hh signalling pathway is functional in the gut, the effect of Shh on epithelial cells in vitro was explored by treating primary murine colonocytes with either Shh peptide or neutralizing anti-Shh antibody. The proportion of cells in the S-phase was assessed by bromodeoxyuridine (BrdU) incorporation. It was found that exogenous Shh promotes cell proliferation in colonocytes, while anti-Shh inhibits proliferation, suggesting that Shh is required during proliferation of epithelial cells in vitro. It is suggested that SHH is required during epithelial proliferation in the colon and that there is a possible role for Hh signalling in epithelial colon tumour progression in vivo.