Post-mortem microbiology in sudden death: sampling protocols proposed in different clinical settings.

BACKGROUND: Autopsies, including minimally invasive autopsies, are a powerful tool for determination of the cause of death. When a patient dies from an infection, microbiology is crucial to identify the causative organism. Post-mortem microbiology (PMM) aims to detect unexpected infections causing sudden deaths; confirm clinically suspected but unproven infection; evaluate the efficacy of antimicrobial therapy; identify emergent pathogens; and recognize medical errors. Additionally, the analysis of the thanatomicrobiome may help to estimate the post-mortem interval. AIMS: The aim was to provide advice in the collection of PMM samples and to propose sampling guidelines for microbiologists advising autopsy pathologists facing different sudden death scenarios. SOURCES: A multidisciplinary team with experts in various fields of microbiology and autopsies on behalf of the ESGFOR (ESCMID - European Society of Clinical Microbiology and Infectious Diseases - study group of forensic and post-mortem microbiology and in collaboration with the European Society of Pathology) developed this narrative review based on a literature search using MedLine and Scopus electronic databases supplemented with their own expertise. CONTENT: These guidelines address measures to prevent sample contamination in autopsy microbiology; general PMM sampling technique; protocols for PMM sampling in different scenarios and using minimally invasive autopsy; and potential use of the evolving post-mortem microbiome to estimate the post-mortem interval. IMPLICATIONS: Adequate sampling is paramount to identify the causative organism. Meaningful interpretation of PMM results requires careful evaluation in the context of clinical history, macroscopic and histological findings. Networking and closer collaboration among microbiologists and autopsy pathologists is vital to maximize the yield of PMM.

Use of gene profiling to evaluate the effects of a feed additive on immune function in periparturient dairy cattle.

Objectives were to investigate mechanisms by which a nutritional supplement alters immunity in dairy cattle. Our hypothesis was that feeding this product to dairy cattle altered neutrophil gene expression. Eight periparturient Jersey cattle were randomly assigned to one of two treatments: control and treated. Control animals were fed a dry cow ration for 1 month prior to calving. The treated cows were fed the same ration supplemented with OmniGen-AF. Following calving, blood samples were taken and neutrophils were prepared after which RNA was extracted. Gene expression in neutrophils of treated versus control-fed animals was then assessed using bovine-total leukocyte (BOTL-5) arrays. Eighteen genes were differentially regulated in the experimental group and of these, twice as many were up-regulated as down-regulated. Patterns of changes indicated that the additive might alter neutrophil apoptosis, signaling and sensitivity. Two of the regulated genes [interleukin-1beta converting enzyme (ICE) and interleukin-4 receptor (IL-4R)] were investigated in more detail using quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR). Each was found to be elevated by the feeding of experimental product. Increased expression of ICE indicates potential for enhanced neutrophil expression of interleukin-1beta (IL-1beta), a cytokine which plays roles in the inflammatory response and which stimulates adaptive immunity following innate immune activation. Altered expression of IL-4R indicates potential for changes in neutrophil apoptosis. The experiment identified mechanisms by which the additive altered neutrophil gene expression. While many nutrients support the immune system, we have shown that a non-traditional nutritional approach may also have utility in modulating immune function.

Microarray analysis reveals difference in gene expression profiles of hair and wool sheep infected with Haemonchus contortus.

Sheep infected with the abomasal parasite, Haemonchus contortus, have reduced growth rates, decreased wool production, and anemia, and heavy infections may result in death. Anthelmintic treatment can remove worms, but the cost of treatment and prevalence of drug-resistant worms has led to greater focus on genetic resistance of the host to parasitism. Variation in parasite resistance exists within and among sheep breeds, and Caribbean hair sheep have greater resistance than most conventional wool breeds. Our objective was to investigate differences in gene expression between 24 parasite-resistant hair and 24 susceptible wool lambs to determine genetic mechanisms involved in resistance to H. contortus. Half of the animals of each breed were infected and sacrificed at 3 or 27 days post-infection; the remaining animals were uninfected controls. Breed differences in abomasum and abomasal lymph node tissue gene expression were assessed using bovine cDNA microarrays. Over 60 transcripts differed between breeds for each tissue and infection status. Genes differentially expressed between hair and wool sheep 3 days PI were assessed for gene function and mechanisms for greater immune cell infiltration, abomasal tissue repair, Th17 response, and anticoagulation were present in parasite-resistant hair sheep. By 27 days PI, hair sheep had greater expression of genes involved in gut motility, inflammatory cytokines, and cell proliferation and differentiation compared to wool sheep. Changes in these processes indicate Caribbean hair sheep have a stronger inflammatory response when infected with H. contortus which may facilitate the increased parasite resistance observed in these sheep.

Transportation of young beef bulls alters circulating physiological parameters that may be effective biomarkers of stress.

Transportation causes stress in cattle that may alter numerous physiological variables with a negative impact on production and health. The objectives of the current study were to investigate the physiological effects of truck transportation and to characterize a pattern of phenotypes in the circulation that may aid in the early identification of stress-susceptible animals that often succumb to severe respiratory disease. Thirty-six young beef bulls (Aberdeen Angus, n = 12; Friesian, n = 12; and Belgian Blue x Friesian, n = 12) were subjected to a 9-h truck transportation by road. Blood (10 mL) was collected at -24, 0, 4.5, 9.75, 14.25, 24, and 48 h relative to the initiation of transportation (0 h). Plasma was collected for the assay of various metabolic, inflammatory, and steroid variables, and total leukocyte counts were determined in whole blood at each time point. Body weight and rectal temperature were recorded at -24, 9.75, and 48 h. Transportation decreased measures of protein metabolism in the plasma, including albumin (P = 0.002), globulin (P < 0.001), urea (P = 0.006), and total protein (P < 0.001), and increased creatine kinase (P < 0.001). The energy substrate beta-hydroxybutyrate was not changed (P = 0.27). Acute phase proteins haptoglobin and fibrinogen were both decreased (P < 0.001), whereas total leukocyte counts were elevated (P = 0.002). Circulating steroid concentrations were altered, because a classical acute increase in plasma cortisol was observed with the onset of transit (P < 0.001), in association with a decrease in dehydroepiandrosterone (P = 0.07), resulting in a profound increase in cortisol:dehydroepiandrosterone ratio (P < 0.001). Plasma testosterone was decreased, whereas plasma progesterone was increased (P < 0.001) in association with the increase in cortisol (P < 0.001). There was also an effect of breed for all variables except plasma urea, creatine kinase, and testosterone, perhaps indicating that a genetic component contributed to the physiological response to transportation stress, although without any clear trend. Taken together, this profile of physiological variables in the circulation of transportation-stressed bulls may aid in the future detection of disease-susceptible cattle after transportation. Further research to validate these potential biomarkers is necessary.

Depressed DHEA and increased sickness response behaviors in lame dairy cows with inflammatory foot lesions.

Lameness is a multifactorial condition influenced by the environment, genetics, management and nutrition. Detection of lameness is subjective and currently limited to visual locomotion observations which lack reliability and sensitivity. The objective of this study was to search for potential biomarkers of inflammatory foot lesions that underlie most cases of lameness in dairy cows, with a focus on the sickness response and relevant endocrine, immune and behavioral changes. Serum and peripheral blood mononuclear cells (PBMC) were collected from eight sound and eight lame high-producing Holstein cows. Immune cell activation was investigated in PBMCs using a candidate gene approach in which the expression of pro-opiomelanocortin, interleukin-1beta, l-selectin, matrix metalloproteinase-9 and glucocorticoid receptor-alpha was measured via quantitative real time-RT-PCR. Endocrine changes were investigated by monitoring serum concentrations of cortisol and dehydroepiandrosterone (DHEA). Additionally, systematic behavioral observations were carried out to characterize a behavioral profile associated with a sickness response typical of this condition. Lame cows showed significantly lower eating (P=0.01) and ruminating (P=0.01) behaviors and higher incidence of self-grooming (P=0.04) compared to sound cows. Lame cows also showed a 23% decrease in serum DHEA (P=0.01) and 65% higher cortisol:DHEA ratio (P=0.06) compared to sound cows. However, no significant differences were found in candidate gene expression between lame and sound cows. In association with sickness behaviors, serum DHEA concentration and cortisol:DHEA ratio are promising objective indicators of inflammatory foot lesions in dairy cattle and may be useful as diagnostic targets for animals in need of treatment.

Transportation stress alters the circulating steroid environment and neutrophil gene expression in beef bulls.

Stress and its association with altered immune function and incidence of respiratory diseases in cattle have lead to concerns over animal health and welfare during truck transportation. Previously, bulls subjected to transportation stress displayed altered expression of candidate neutrophil genes, warranting a broader investigation of the neutrophil transcriptome and possible associations with fluctuations in circulating steroid hormones. In the current study, blood was collected from six Belgian BluexFriesian bulls at -24, 0, 4.5, 9.75, 14.25, 24, and 48h relative to initiation of 9h of truck transportation. Plasma concentrations of cortisol, dehydroepiandrosterone (DHEA), progesterone, and testosterone were measured; cortisol:DHEA ratios were computed. Neutrophil gene expression was monitored by microarray analysis using bovine immunobiology (BOTL-5) microarrays. Eighty-eight genes were identified as being differentially expressed at P<0.05. Putatively affected genes were grouped into ontological clusters; those of greatest interest for qRT-PCR validation were involved in immune response, apoptosis, wound healing, and several of currently unknown function. Confirmed gene expression changes supported the dramatic effects of transportation stress on the bovine neutrophil transcriptome. Temporal correlations between gene expression profiles and circulating total leukocyte and neutrophil counts were apparent. However, few relationships between gene expression and plasma steroid profiles were detected, possibly due to the biological time-lag between these variables not captured by the blood collection schedule. Further investigation into the factors underlying neutrophil gene expression changes and validations at the protein and cell behavior levels will lead to a better understanding of altered innate immunity in cattle during transportation stress.

Transportation stress in young bulls alters expression of neutrophil genes important for the regulation of apoptosis, tissue remodeling, margination, and anti-bacterial function.

The transportation of beef cattle results in a stress response that is associated with increased susceptibility and severity of respiratory diseases, presumably due to an alteration in immune function. Neutrophils are phagocytic immune cells important in lung defense and are also targets of the stress response. The objective of this study was to determine if a 9h transportation of young bulls by road induced changes in the expression of candidate genes known to be important in neutrophil-mediated defense and inflammation in the lung. These neutrophil genes encompassed functions of apoptosis (A1 and Fas), tissue remodeling (MMP-9), vascular margination (L-selectin), bacterial killing (BPI), and wound healing (betaglycan), as well as responsiveness of the cells to stress-induced increases in glucocorticoid hormones (GRalpha). To explore gene expression changes, blood was collected, plasma harvested, and neutrophils isolated from six Belgian Blue x Friesian bulls (231+/-7.0 kg in weight; 282+/-4 days of age) at -24, 0, 4.5, 9.75, 14.25, 24, and 48h relative to commencement of a 9h road transportation by truck. Plasma cortisol concentrations were elevated at 4.5 and 9.75h, peaking at 50.64+/-4.46 ng/mL (P<0.0001) and confirming that the animals experienced stress. Blood neutrophil count was elevated between 4.5 and 14.25h (P<0.0001), reaching a peak that was over 3-fold higher than the -24h concentration. Neutrophil Fas gene expression was acutely down-regulated (P=0.02) by transportation stress, while expressions of MMP-9, l-selectin, and BPI were profoundly up-regulated (P=0.003, 0.002, and <0.001 respectively). However, no changes in neutrophil expressions of betaglycan, GRalpha, and A1 were detected. It is concluded that a 9h transportation of young bulls induces a gene expression signature in blood neutrophils that increases their circulating numbers and may enhance their pro-inflammatory and anti-bacterial potential.

Ability of a commercial feed additive to modulate expression of innate immunity in sheep immunosuppressed with dexamethasone.

In the first study, we tested the ability of a commercial feed additive (OmniGen-AF) to affect markers of innate immunity in immunosuppressed sheep and the ability of a pathogen challenge (mould) to affect the immune response to the additive. Treatments consisted of (1) control, (2) immunosuppressed with dexamethasone (DEX), (3) immunosuppressed plus the feed additive, (4) immunosuppressed plus Aspergillus fumigatus and (5) immunosuppressed, A. fumigatus and the additive. Animal health was monitored and indexes of innate immunity (neutrophil L-selectin and interleukin-1ß (IL-1ß)) were collected. DEX caused immunosuppression (i.e. reduced abundance of neutrophil L-selectin and IL-1ß). This immunosuppressive effect was countered by the provision of the additive in the ration. Provision of mould in the ration increased the ability of the additive to regulate markers of innate immune function. A second study was completed to re-assess the properties of the additive and other feed products. The study consisted of seven treatments: (1) immunosuppressed, (2) immunosuppressed with additive, (3) immunosuppressed with additive in pelleted form (low-temperature pellet) and (4) immunosuppressed with additive in a high-temperature pellet. The remaining three treatments assessed abilities of three other additives to regulate markers of innate immune function. In this study, OmniGen-AF increased expression of neutrophil L-selectin abundance in immunosuppressed animals and this was unaffected by the pelleting temperature. None of the other additives affected markers of innate immunity. In these studies we discovered mechanisms by which a feed product may affect the immune function of ruminant livestock. The product countered DEX-dependent down-regulation of markers of innate immune function and its actions were enhanced by the presence of pathogen (mould) in the ration.

Bee pollen product supplementation to horses in training seems to improve feed intake: A pilot study.

The objective of this study was to determine the efficacy of supplementation of Dynamic Trio 50/50, a bee pollen-based product, to improve physical fitness, blood leukocyte profiles, and nutritional variables in exercised horses. Ten Arabian horses underwent a standardised exercise test (SET), then were pair-matched by sex and fitness and randomly assigned to BP (receiving 118 g of Dynamic Trio 50/50 daily) or CO (receiving 73 g of a placebo) for a period of 42 days. A total collection was conducted from days 18 to 21 on six geldings to determine nutrient retention and neutral detergent fibre (NDF) and acid detergent fibre (ADF) digestibility. Horses were exercise conditioned and completed another SET on day 42. V160 and V200 were calculated from SET heart rates (HR). Lactate, glucose, haematocrit (HT) and haemoglobin (HB) concentrations were determined from SET blood samples. Total leukocyte count, and circulating numbers of various leukocytes and IgG, IgM and IgA concentrations were determined in rest and recovery blood samples from both SETs. Geldings on BP (n = 3) ate more feed than CO. BP had less phosphorus excretion, and tended to retain more nitrogen. BP tended to digest more NDF and ADF while having lower NDF digestibility and tending to have lower ADF digestibility. No treatment differences existed for V160 and V200, HR, lactate, HT and HB. There was a trend for lymphocyte counts to be lower in BP than CO on day 42. Dynamic Trio 50/50 supplementation may have a positive effect on performance by helping horses in training meet their potentially increased nutrient demands by increasing feed intake and thus nutrient retention.

Analysis of the bovine neutrophil transcriptome during glucocorticoid treatment.

The objective of this study was to characterize a large portion of the bovine neutrophil transcriptome following treatment with the anti-inflammatory glucocorticoid dexamethasone (Dex). Total RNA was isolated from blood neutrophils of healthy cattle (5 castrated male Holsteins) immediately following cell purification (0 h) or after ex vivo aging for 4 h with or without added Dex. Additional neutrophils were cotreated with a glucocorticoid receptor (GR) antagonist (RU486) and Dex for 4 h. RNA was amplified, dye labeled (Cy3 or Cy5), and hybridized to a series of National Bovine Functional Genomics Consortium (NBFGC) microarrays. LOWESS data normalization followed by mixture model analyses showed that 11.15% of the spotted NBFGC cDNAs (2,036/18,263) were expressed in 4-h (untreated) neutrophils. Subsequent two-step mixed-model analysis detected (P < or = 0.05) 1,109 differentially expressed genes, of which contrast analysis indicated those that were independently responsive to aging (1,064), Dex (502), RU486 + Dex (141), or RU486 (357). In silico analysis revealed that 416 of the differentially expressed genes are unknown, 59 did not cluster well based on known function, and 634 clustered into 20 ontological categories. Independent validation of differential expression was done for 14 of the putatively Dex-responsive genes across these categories. Results showed that Dex induced rapid translocation of GR into the neutrophil nucleus and signaled dramatic alterations in expression of genes that delay apoptosis, enhance bactericidal activity, and promote tissue remodeling without inflammation or fibrosis. Thus these findings revealed hitherto unappreciated plasticity of blood neutrophils and potentially novel anti-inflammatory/wound-healing actions of glucocorticoids.

Short communication: A potential antiapoptotic phenotype in neutrophils of cows milked once daily in early lactation.

The objectives of this experiment were to compare the circulating concentrations of cortisol and determine whether these correlated with the expression profiles of a set of candidate apoptosis genes in neutrophils of Holstein-Friesian cows milked once vs. 3 times daily for 28 d postpartum. Cows on the once-daily milking regimen had significantly higher plasma cortisol concentrations on d 3, 14, and 28 postpartum than did those milked 3 times daily. On d 3 postpartum, when differences in cortisol and neutrophil counts were highest between the groups, mean mRNA abundance of nuclear factor kappaB p65 subunit, IkappaBalpha, X-linked inhibitor of apoptosis; and heat shock protein 70 were higher in neutrophils of the cows milked once daily than in cows milked 3 times daily. However, no correlations were detected among plasma cortisol concentration, neutrophil count, or neutrophil gene expression in this study. Results suggest that the modest neutrophilia associated with once daily milking of cows immediately postpartum may be related to modifications in the cells' apoptotic program by factors other than cortisol.

Genetic basis and risk factors for infectious and noninfectious diseases in US Holsteins. I. Estimation of genetic parameters for single diseases and general health.

Health data collected from 1996 to 1999 from 177 herds in Minnesota and Wisconsin were analyzed to establish genetic basis for infectious and noninfectious diseases. Three types of health traits were targeted. First, available infectious conditions were used to identify animals that are superior in their general immunity (including innate immunity) for infectious diseases. Generalized immunity may be thought of as a combination of immune responses to a variety of immune system challenges. Second, single infectious and noninfectious diseases were analyzed separately. Third, infectious reproductive diseases as one category of related conditions, and cystic ovary disease as one category of 3 related noninfectious ovary disorders were studied. Data were analyzed using a threshold model that included herd, calving year, season of calving, and parity as cross-classified fixed factors; and sire and cow within sires as random effects. Days at risk and days in milk at the beginning of a record were included by fitting the days as continuous covariates in the model. A heritability value of 0.202 +/- 0.083 was estimated for generalized immunity. Heritability values of 0.141 and 0.161 were estimated for uterine infection and mastitis, respectively. Heritability of single noninfectious disorders ranged from 0.087 to 0.349. The amount of additive genetic variance recovered in the underlying scale of noninfectious disorders tended to zero when combining multiple conditions. The study supports combining infectious diseases into categories of interest but we do not recommend the same approach for noninfectious disorders.

Genetic variation in bovine mononuclear leukocyte responses to dexamethasone.

The objective of this study was to determine whether bovine mononuclear leukocytes exhibit genetic variability prior to and after a glucocorticoid hormone challenge in vivo. Test animals included 60 pedigreed Holstein bulls treated on 3 consecutive days with dexamethasone and 5 untreated control bulls. Eight indicator traits of leukocyte responsiveness to dexamethasone included the percentages of circulating B cells, T cells (CD4, CD8, and workshop cluster 1 molecule expressed by bovine gammadelta T cell), major histocompatibility complex (MHC) I and II expressing cells, and mean expressions of surface MHC I and MHC II on circulating cells. Blood for this work was collected from each test bull 10 times before, during, and after dexamethasone administration, with corresponding samples taken for control bulls. Random regression models with treatment-specific serial correlation were applied to the leukocyte data sets to estimate genetic and nongenetic sources of variation in baseline and recovery aspects of the traits. All traits responded predictably to glucocorticoid challenge. Genetic variation was observed in baseline measurements of all traits, with heritability estimates ranging from 0.21 +/- 0.03 to 0.60 +/- 0.06. Genetic variation in linear recovery from nadir values following dexamethasone administration was significant only for percentage CD4, percentage CD8, and for surface expression of MHC II. The genetic covariance between basal and linear recovery was positive and significant for percentage CD4, percentage CD8, and MHC II expression. The bovine lymphocyte antigen DRB3.2 locus accounted for significant proportions of total variation in percentage MHC II cells and MHC I expression. These results suggest that genetic variability exists for important basal and glucocorticoid-modified phenotypes of bovine mononuclear leukocytes, implying that immunocompetence traits impacted by this stress hormone may be enhanced by genetic selection.

Short communication: amino acid transporters in porcine mammary gland during lactation.

The objective of this study was to determine whether mRNA transcripts for the amino acid (AA) transporter proteins CAT-1, CAT-2B, B(0,+), and ASCT1 are present in porcine mammary tissue (MT). Transcript abundance on d 7 and 17 of lactation was determined by Northern blot analysis, and absolute quantification was performed by real-time polymerase chain reaction. Porcine MT expresses CAT-1, CAT-2B, B(0,+), and ASCT1 during lactation. Preliminary findings indicate that B(0,+) mRNA abundance tended to decrease on d 17 compared with that on d 7.

Hyperimmunization of steers with J5 Escherichia coli bacterin: effects on isotype-specific serum antibody responses and cross reactivity with heterogeneous gram-negative bacteria.

Isotype-specific antibody responses and cross reactivity were profiled following hyperimmunization of steers with J5 Escherichia coli bacterin. The vaccine was administered at time 0, 30 d later, and every 2 wk for 10 subsequent immunizations. Blood was collected preimmunization and multiple times following each immunization. Isotype-specific anti-J5 Escherichia coli antibody response profiles in diluted sera harvested from each sample were assayed by ELISA and recorded as optical density. Selected sera were assayed for anti-J5 Escherichia coli antibody titers and used to determine cross reactivity against a variety of gram-negative bacteria. Immunization number and day postimmunization influenced response profiles for anti-J5 E. coli IgM, IgG(1)and IgG(2) antibodies. Two immunizations increased mean serum IgM and the IgG(1)antibody profiles above preimmunization levels, but 5 immunizations were required to detect significant IgG(2) antibody responses that were above preimmunization levels. Isotype-specific cross reactivity of the serum antibodies with a variety of heterologous gram-negative bacteria was also increased by hyperimmunization. However, no cross reactivity was observed for Staphylococcus aureus, purified lipopolysaccharide, or lipid A. Our results indicate that multiple booster doses of J5 E. coli bacterin may be required to elicit high levels of cross-reactive serum IgG(2) antibodies.

Videos have a role in postgraduate necropsy education.

AIMS: This is the first study to investigate the usefulness of structured, scripted videos as an adjunct to the mortuary based training of histopathology trainees in necropsy techniques. METHODS: Four structured and scripted videos describing aspects of necropsy health and safety, evisceration, general dissection techniques, specialist dissection techniques, and reconstruction were shown to histopathology trainees attending the 2001 University of Sheffield short course on the autopsy. Delegates who agreed to participate in the study were asked to complete a short questionnaire seeking Likert-type and free text responses concerning the usefulness of the videos in postgraduate necropsy training. Free text responses were analysed using a themed content analysis. RESULTS: All 38 delegates who viewed the videos agreed to participate in the study. Of these, 35 found the videos enjoyable and 34 found them interesting. Thirty one felt the videos enhanced their learning experience. Advantages of the videos included the ability to learn about specialist techniques rarely encountered in the mortuary, the ability to teach large numbers of students at once, allowing students to learn at their own pace, and as a tool for revision. Repetition between the videos, a lack of interactivity, and a lack of sufficient detail on general necropsy techniques were felt by participants to be the principal disadvantages of this teaching tool. CONCLUSIONS: Videos are an acceptable teaching tool for students. They have a valuable role to play as an adjunct to dissection in teaching junior histopathology trainees about specialist necropsy dissection techniques.

Multivariate head injury threshold measures for various sized children seated behind vehicle seats in rear impacts.

Government recommendations to place children into the rear areas of motor vehicles to avoid airbag induced injuries have been complicated by the fact that most adult occupied front seats will collapse into the rear area during rear-impacts, and thus pose another potentially serious injury hazard to rear-seated children. Many variables affect whether or not a front seat occupant will collapse into the rear child, and whether that interaction could be injurious to the child. For instance, the severity of rear impact, coupled with front and rear occupant sizes (mass and stature), and the level of front seat strength, all interrelate to influence whether or not a rear seated child is likely to be impacted and possibly injured. The most common types of child injuries in these instances are head and chest injuries. In this study, a "high-low" experimental method was employed with a multi-level "factorial analysis" technique to study "multivariate" biomechanics of child head injury potential determined from rear-seated 3 and 6 year-old child surrogates in different types of vehicle bodies mounted to a sled system. The sled-buck systems were towed rearward into crushable barriers that matched the crash pulses of the vehicle types being tested. Various sizes of adult surrogates (i.e. 50 kg up to 110 kg), seated in both the "typical" low strength "single recliner" collapsing type front seat (i.e. 3.2 kN) and a much stronger "belt-integrated" seat design (i.e. up to 14.5 kN), were tested in the two different "sled body-buck" set-ups at various impact levels (i.e. 22.5 to 50 kph). One set-up used a popular minivan vehicle body with "built-in booster" seats for the 3 year-old. The other used a 4-door family sedan vehicle body with the 6 year-old in a standard rear bench seat. The parameters of the tests enabled the experimental data to be combined into polynomial "head injury" functions of the independent variables so the "likelihood" of rear child head-injury potential could be "mapped" over ranges of the key parameters. Accident cases were compared with predictions to verify the methodology.

Medical educators' personal attitudes towards the necropsy.

AIMS: To explore the personal attitudes of medical educators to the necropsy. METHODS: A "theoretical sample" of individuals, with widely disparate views, was selected from University of Sheffield Medical School affiliated staff. These individuals underwent semi-structured interviews (in private) to elucidate their personal attitudes towards the necropsy (in relation to the possibility of their own necropsy or that of a close relative). RESULTS: Nine themes pertaining to personal attitudes towards the necropsy were identified. In general medical educators had a positive attitude towards the necropsy. CONCLUSIONS: This study suggests that medical educators still believe that the necropsy is of value in medical education, despite dramatic curricular revisions in the UK, declining necropsy rates, and adverse media attention to the necropsy.

Refining the diagnosis of hydatidiform mole: image ploidy analysis and p57KIP2 immunohistochemistry.

AIM: To determine whether image analysis of ploidy status and immunohistochemical analysis of p57KIP2 (a paternally imprinted, maternally expressed gene) can be used to refine the diagnosis of molar pregnancy. METHODS AND RESULTS: The original histological diagnosis in 40 randomly selected cases of hydatidiform mole was reviewed and confirmed in 38 cases (22 complete moles, 16 partial moles). These cases were anonymized and submitted for further analysis. Tissue from each case was submitted for flow cytometric assessment of DNA ploidy using a FACSort flow cytometer and for automated image cytometric assessment using a novel digital imaging system. Tissue sections from each case were immunostained with a monoclonal mouse antibody to p57KIP2. Correlations between the histopathological diagnosis, image cytometry, flow cytometry and p57KIP2 immunohistochemistry were determined using kappa statistics. The concordance between histological diagnosis and p57KIP2 was very good (kappa = 0.89). Twenty of the 22 (90.9%) complete moles showed no immunoreactivity for p57KIP2. The remaining two cases showed nuclear immunoreactivity in villous cytotrophoblast. In one of these, the pattern of staining resembled that of a partial mole. In the other, the staining pattern supported the diagnosis of a twin molar/non-molar pregnancy. All 16 partial moles were p57KIP2 immunoreactive. On flow cytometry, all 22 complete moles were diploid and 12/16 partial moles were triploid (the remaining four cases originally diagnosed as partial moles were found to be diploid). On image cytometry, one case originally diagnosed as complete mole was found to contain a triploid population. Thus, by using a combination of image cytometry and p57KIP2 status we were able to refine the diagnosis of molar pregnancy in five (13%) of the cases studied. CONCLUSIONS: Automated image cytometry is a readily performed investigation which is comparable to, but more sensitive than, flow cytometry. Complementary use of ploidy analysis and p57KIP2 status can now help to distinguish a diploid hydropic miscarriage (p57KIP2-positive), diploid complete mole (p57KIP2-negative) and triploid partial mole (p57KIP2-positive).

Necropsy practice after the "organ retention scandal": requests, performance, and tissue retention.

AIMS: After the so called "organ retention scandal" in the UK this study set out to assess the impact on death certification and hospital (consent) necropsies, including the postmortem retention of tissues and organs. METHODS: Data were prospectively gathered over a one year period for all deaths occurring at the Royal Hallamshire Hospital, Sheffield, UK to determine the frequencies with which death certificates were completed and necropsies were requested. The seniority of the clinician undertaking these duties was recorded. Pathologists were asked to record the extent of every necropsy during the study period. The type and planned uses of tissues retained were recorded. RESULTS: Death certificates were issued for 88.5% of the 966 deaths for which clinicians completed proformas. Of these, 88.9% were issued by preregistration and senior house officers. Consent was sought for a necropsy in 6.2% of cases (usually by non-consultant staff) and was granted in 43.4% of these. The overall, medicolegal, and hospital necropsy rates were 13.4%, 9.9%, and 3.5%, respectively. Tissues were retained from 55.4% of necropsies for diagnostic purposes, although sampling does not appear to be systematic. CONCLUSIONS: Death certification and seeking consent for a necropsy are frequently delegated to junior clinical staff. This may explain the low standard of death certification reported by others and the low necropsy rate. The decline in the necropsy rate and the low rate of sampling for histological examination highlight the decline of the hospital necropsy and the lack of a systematic approach to tissue sampling.