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Host and pathogen factors that influence variability of Mycobacterium tuberculosis lipid body content in sputum from patients with tuberculosis: an observational studyBackground: High proportions of Mycobacterium tuberculosis cells in sputum containing triacylglycerol-rich lipid bodies have been shown to be associated with treatment failure or relapse following antituberculous chemotherapy. Although lipid body determination is a potential biomarker for supporting clinical trial and treatment decisions, factors influencing variability in sputum frequencies of lipid body-positive (%LB+) M tuberculosis in patients are unknown. We aimed to test our hypothesis that exposure to host-generated NO and M tuberculosis strains are factors associated with differences in sputum %LB+. Methods: In this observational study, we determined %LB+ frequencies before treatment by microscopy in patients with smear-positive tuberculosis from two separate prospective observational study settings (Gondar, Ethiopia, recruited between May 1, 2010, and April 30, 2011, and Fajara, The Gambia, who provided sputum samples before treatment between May 5, 2010, and Dec 22, 2011). In Ethiopia, fractional exhaled nitric oxide (FeNO) was measured as a biomarker of host NO, and M tuberculosis strain differences were determined by spoligotyping. Treatment response was assessed by percentage weight change after 7 months. In The Gambia, treatment responses were assessed as change in BMI and radiographic burden of disease after 6 months. Sputum M tuberculosis isolates were studied in vitro for their %LB+ and triacylglycerol synthase 1 (tgs1) mRNA responses to NO exposure. Propidium iodide staining was used as a measure of NO strain toxicity. Correlation between in vitro %LB+ frequencies following NO exposure and those of the same strain in sputum was examined with linear regression and Dunnett's multiple comparison test. Findings: In Ethiopia, 73 patients who were smear positive for pulmonary tuberculosis were recruited (43 [59%] were male and 30 [41%] were female). Of these, the %LB+ in the sputum of 59 patients showed linear correlation with log10 FeNO (r2=0·28; p<0·0001) and an association with strain spoligotype was suggested. Seven M tuberculosis strains from The Gambia showed different dose-responses to NO in vitro, demonstrated by changing lipid body content, tgs1 transcription, and bacterial toxicity. In sputum %LB+ frequencies correlated with in vitro %LB+ responses to NO of the corresponding isolate. In a subset of 34 patients across both cohorts, higher sputum %LB+ frequencies before treatment were associated with weaker responses to treatment than lower sputum %LB+ frequencies. Interpretation: M tuberculosis strain and exposure to host-generated NO are associated with sputum %LB+. Our results support the use of M tuberculosis strain-dependent sputum %LB+ as a predictive biomarker of treatment response. Funding: The Medical Research Council, the University of Leicester, and the University of Gondar.
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An optimised patient-derived explant platform for breast cancer reflects clinical responses to chemotherapy and antibody-directed therapyBreast Cancer is the most common cancer among women globally. Despite significant improvements in overall survival, many tumours are refractory to therapy and so novel approaches are required to improve patient outcomes. We have evaluated patient-derived explants (PDEs) as a novel preclinical platform for breast cancer (BC) and implemented cutting-edge digital pathology and multi-immunofluorescent approaches for investigating biomarker changes in both tumour and stromal areas at endpoint. Short-term culture of intact fragments of BCs as PDEs retained an intact immune microenvironment, and tumour architecture was augmented by the inclusion of autologous serum in the culture media. Cell death/proliferation responses to FET chemotherapy in BC-PDEs correlated significantly with BC patient progression-free survival (p = 0.012 and p = 0.0041, respectively) and cell death responses to the HER2 antibody therapy trastuzumab correlated significantly with HER2 status (p = 0.018). These studies show that the PDE platform combined with digital pathology is a robust preclinical approach for informing clinical responses to chemotherapy and antibody-directed therapies in breast cancer. Furthermore, since BC-PDEs retain an intact tumour architecture over the short-term, they facilitate the preclinical testing of anti-cancer agents targeting the tumour microenvironment.
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The 1000 Mitoses Project: a consensus-based international collaborative study on mitotic figures classificationIntroduction. The identification of mitotic figures is essential for the diagnosis, grading, and classification of various different tumors. Despite its importance, there is a paucity of literature reporting the consistency in interpreting mitotic figures among pathologists. This study leverages publicly accessible datasets and social media to recruit an international group of pathologists to score an image database of more than 1000 mitotic figures collectively. Materials and Methods. Pathologists were instructed to randomly select a digital slide from The Cancer Genome Atlas (TCGA) datasets and annotate 10-20 mitotic figures within a 2 mm2 area. The first 1010 submitted mitotic figures were used to create an image dataset, with each figure transformed into an individual tile at 40x magnification. The dataset was redistributed to all pathologists to review and determine whether each tile constituted a mitotic figure. Results. Overall pathologists had a median agreement rate of 80.2% (range 42.0%-95.7%). Individual mitotic figure tiles had a median agreement rate of 87.1% and a fair inter-rater agreement across all tiles (kappa = 0.284). Mitotic figures in prometaphase had lower percentage agreement rates compared to other phases of mitosis. Conclusion. This dataset stands as the largest international consensus study for mitotic figures to date and can be utilized as a training set for future studies. The agreement range reflects a spectrum of criteria that pathologists use to decide what constitutes a mitotic figure, which may have potential implications in tumor diagnostics and clinical management.
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Broad evidence of xylazine in the UK illicit drug market beyond heroin supplies: Triangulating from toxicology, drug-testing and law enforcementBackground and aims: Xylazine is a non-opioid sedative which has spread rapidly throughout the US illicit drug supply. This study aimed to describe the spread of xylazine throughout the UK illicit drug supply. Methods: Xylazine detections in human biological samples were collated from toxicology laboratories operating in the United Kingdom with the date, location, case type, xylazine concentration and co-detected drugs (with quantifications where performed) detailed, where permitted, by the corresponding coroner. Drug-testing cases positive for xylazine were collated from the Welsh Emerging Drugs and Identification of Novel Substances (WEDINOS) drug-testing postal service with the date, location, purchase intent and co-detected drugs detailed. Drug seizures made by UK law enforcement were communicated by the Office for Health Improvement and Disparities with the date and location detailed. Results: By the end of August 2023, xylazine was detected in 35 cases from throughout toxicology, drug-testing and drug seizure sources covering England, Scotland and Wales. There were no cases reported from Northern Ireland. Xylazine was detected in biological samples from 16 people. In most cases where full toxicology results were provided, xylazine was detected with heroin and/or a strong opioid (n = nine of 11), but this polydrug use pattern was not evident in all cases (n = two of 11), suggesting a wider circulation of xylazine in the UK illicit drug market beyond heroin supplies. Evidence from WEDINOS supports this claim, as all 14 drug samples (100%) submitted from across the UK contained xylazine; however, in none of these cases was heroin the purchase intent but rather counterfeit prescription medication tablets (n = 11 of 14), tetrahydrocannabinol (THC) vapes (n = two of 14) or white powder (n = one of 14). Additional evidence for the spread of illicit xylazine comes from five drug seizures made by law enforcement. Conclusions: Xylazine has penetrated the UK illicit drug market and is not limited to heroin supplies.
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A scoping review of artificial intelligence in medical education: BEME Guide No. 84Background: Artificial Intelligence (AI) is rapidly transforming healthcare, and there is a critical need for a nuanced understanding of how AI is reshaping teaching, learning, and educational practice in medical education. This review aimed to map the literature regarding AI applications in medical education, core areas of findings, potential candidates for formal systematic review and gaps for future research. Methods: This rapid scoping review, conducted over 16 weeks, employed Arksey and O'Malley's framework and adhered to STORIES and BEME guidelines. A systematic and comprehensive search across PubMed/MEDLINE, EMBASE, and MedEdPublish was conducted without date or language restrictions. Publications included in the review spanned undergraduate, graduate, and continuing medical education, encompassing both original studies and perspective pieces. Data were charted by multiple author pairs and synthesized into various thematic maps and charts, ensuring a broad and detailed representation of the current landscape. Results: The review synthesized 278 publications, with a majority (68%) from North American and European regions. The studies covered diverse AI applications in medical education, such as AI for admissions, teaching, assessment, and clinical reasoning. The review highlighted AI's varied roles, from augmenting traditional educational methods to introducing innovative practices, and underscores the urgent need for ethical guidelines in AI's application in medical education. Conclusion: The current literature has been charted. The findings underscore the need for ongoing research to explore uncharted areas and address potential risks associated with AI use in medical education. This work serves as a foundational resource for educators, policymakers, and researchers in navigating AI's evolving role in medical education. A framework to support future high utility reporting is proposed, the FACETS framework.
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Digital pathology for reporting histopathology samples, including cancer screening samples - definitive evidence from a multisite studyAims: To conduct a definitive multicentre comparison of digital pathology (DP) with light microscopy (LM) for reporting histopathology slides including breast and bowel cancer screening samples. Methods: A total of 2024 cases (608 breast, 607 GI, 609 skin, 200 renal) were studied, including 207 breast and 250 bowel cancer screening samples. Cases were examined by four pathologists (16 study pathologists across the four speciality groups), using both LM and DP, with the order randomly assigned and 6 weeks between viewings. Reports were compared for clinical management concordance (CMC), meaning identical diagnoses plus differences which do not affect patient management. Percentage CMCs were computed using logistic regression models with crossed random-effects terms for case and pathologist. The obtained percentage CMCs were referenced to 98.3% calculated from previous studies. Results: For all cases LM versus DP comparisons showed the CMC rates were 99.95% [95% confidence interval (CI) = 99.90-99.97] and 98.96 (95% CI = 98.42-99.32) for cancer screening samples. In speciality groups CMC for LM versus DP showed: breast 99.40% (99.06-99.62) overall and 96.27% (94.63-97.43) for cancer screening samples; [gastrointestinal (GI) = 99.96% (99.89-99.99)] overall and 99.93% (99.68-99.98) for bowel cancer screening samples; skin 99.99% (99.92-100.0); renal 99.99% (99.57-100.0). Analysis of clinically significant differences revealed discrepancies in areas where interobserver variability is known to be high, in reads performed with both modalities and without apparent trends to either. Conclusions: Comparing LM and DP CMC, overall rates exceed the reference 98.3%, providing compelling evidence that pathologists provide equivalent results for both routine and cancer screening samples irrespective of the modality used.
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Redox modulation of oxidatively-induced DNA damage by ascorbate enhances both in vitro and ex-vivo DNA damage formation and cell death in melanoma cellsElevated genomic instability in cancer cells suggests a possible model-scenario for their selective killing via the therapeutic delivery of well-defined levels of further DNA damage. To examine this scenario, this study investigated the potential for redox modulation of oxidatively-induced DNA damage by ascorbate in malignant melanoma (MM) cancer cells, to selectively enhance both DNA damage and MM cell killing. DNA damage was assessed by Comet and ɣH2AX assays, intracellular oxidising species by dichlorofluorescein fluorescence, a key antioxidant enzymatic defence by assessment of catalase activity and cell survival was determined by clonogenic assay. Comet revealed that MM cells had higher endogenous DNA damage levels than normal keratinocytes (HaCaT cells); this correlated MM cells having higher intracellular oxidising species and lower catalase activity, and ranked with MM cell melanin pigmentation. Comet also showed MM cells more sensitive towards the DNA damaging effects of exogenous H2O2, and that ascorbate further enhanced this H2O2-induced damage in MM cells; again, with MM cell sensitivity to induced damage ranking with degree of cell pigmentation. Furthermore, cell survival data indicated that ascorbate enhanced H2O2-induced clonogenic cell death selectively in MM cells whilst protecting HaCaT cells. Finally, we show that ascorbate serves to enhance the oxidising effects of the MM therapeutic drug Elesclomol in both established MM cells in vitro and primary cell cultures ex vivo. Together, these results suggest that ascorbate selectively enhances DNA damage and cell-killing in MM cells. This raises the option of incorporating ascorbate into clinical oxidative therapies to treat MM.
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Macroscopy of specimens from the genitourinary systemMacroscopic specimen examination is often critical for accurate histopathology reporting but has generally received insufficient attention and may be delegated to inexperienced staff with limited guidance and supervision. This review discusses issues around macroscopic examination of some common urological specimens; highlighting findings that are critical for patient management and others that are clinically irrelevant. Macroscopic findings are of limited value in completely submitted radical prostatectomy specimens but may be critical in orchidectomy specimens where identification of focal non-seminomatous components can significantly impact patient management. The maximum tumour dimension is often an important prognostic indicator, but specimen dimensions are generally of little clinical utility. Specimens should be carefully examined and judiciously sampled to identify clinically important focal abnormalities such as sarcomatoid change in a renal cell carcinoma and a minor non-seminomatous component in a predominant testicular seminoma. Meticulous macroscopic examination is key as less than 0.2% of the specimen (or macroscopically abnormal area) would be histologically examined even if the entire specimen/abnormal area is submitted for microscopic examination. Retroperitoneal pelvic lymph node dissection specimens for testicular cancer must be handled very differently from other lymph nodal block dissections. Current sampling protocols for transurethral resection of prostate specimens that are based on pre-MRI era data need to be reconsidered because they were specifically designed to detect occult prostate cancer, which would amount to histological cancer screening. Prostatic sampling of cystoprostatectomy specimens should be directed at accurately staging the known bladder cancer rather than detection of incidental prostate cancer.
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A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen testObjectives: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. Methods: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. Results: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). Conclusions: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.
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Identification and characterisation of a rare MTTP variant underlying hereditary non-alcoholic fatty liver diseaseBackground & aims: Non-alcoholic fatty liver disease (NAFLD) is a complex trait with an estimated prevalence of 25% globally. We aimed to identify the genetic variant underlying a four-generation family with progressive NAFLD leading to cirrhosis, decompensation, and development of hepatocellular carcinoma in the absence of common risk factors such as obesity and type 2 diabetes. Methods: Exome sequencing and genome comparisons were used to identify the likely causal variant. We extensively characterised the clinical phenotype and post-prandial metabolic responses of family members with the identified novel variant in comparison with healthy non-carriers and wild-type patients with NAFLD. Variant-expressing hepatocyte-like cells (HLCs) were derived from human-induced pluripotent stem cells generated from homozygous donor skin fibroblasts and restored to wild-type using CRISPR-Cas9. The phenotype was assessed using imaging, targeted RNA analysis, and molecular expression arrays. Results: We identified a rare causal variant c.1691T>C p.I564T (rs745447480) in MTTP, encoding microsomal triglyceride transfer protein (MTP), associated with progressive NAFLD, unrelated to metabolic syndrome and without characteristic features of abetalipoproteinaemia. HLCs derived from a homozygote donor had significantly lower MTP activity and lower lipoprotein ApoB secretion than wild-type cells, while having similar levels of MTP mRNA and protein. Cytoplasmic triglyceride accumulation in HLCs triggered endoplasmic reticulum stress, secretion of pro-inflammatory mediators, and production of reactive oxygen species. Conclusions: We have identified and characterised a rare causal variant in MTTP, and homozygosity for MTTP p.I564T is associated with progressive NAFLD without any other manifestations of abetalipoproteinaemia. Our findings provide insights into mechanisms driving progressive NAFLD. Impact and implications: A rare genetic variant in the gene MTTP has been identified as responsible for the development of severe non-alcoholic fatty liver disease in a four-generation family with no typical disease risk factors. A cell line culture created harbouring this variant gene was characterised to understand how this genetic variation leads to a defect in liver cells, which results in accumulation of fat and processes that promote disease. This is now a useful model for studying the disease pathways and to discover new ways to treat common types of fatty liver disease.
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Macroscopic examination of pathology specimens: a critical reappraisalMeticulous macroscopic examination of specimens and tissue sampling are crucial for accurate histopathology reporting. However, macroscopy has generally received less attention than microscopy and may be delegated to relatively inexperienced practitioners with limited guidance and supervision. This introductory paper in the minisymposium, Macroscopy Under the Microscope, focuses on issues regarding macroscopic examination and tissue sampling that have been insufficiently addressed in the published literature. It highlights the importance of specimen examination and sampling, discusses some general principles, outlines challenges and suggests potential solutions. It is critical to get macroscopy right the first time as it may not be possible to rectify errors even with expert histological assessment or to retrospectively collect missing data after the specimen retention period. Dissectors must, therefore, receive adequate guidance and supervision until they are proficient in macroscopic specimen examination. We emphasise the importance of the clinical context, optimal specimen fixation, succinct and clinically relevant macroscopic descriptions, macrophotography and judicious tissue sampling. We note that current recommendations based on the number of blocks to be submitted per maximum tumour dimension are ambiguous as the amount of tissue submitted in a cassette is not standardised and it is unclear whether 'block' refers to a tissue block or a paraffin block. Concerns around potential oversampling of 'therapeutic' specimens that could result in overdiagnosis due to detection of incidentalomas are also discussed. We hope that the issues discussed in this paper will engender debate on this clinically critical aspect of pathology practice.
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Post mortem blood bromazolam concentrations and co-findings in 96 coronial cases within England and WalesBromazolam is a newly emerging benzodiazepine drug which is not licensed for medicinal use. It may be sourced as a New Psychoactive Substance (NPS) for its desired effects or be consumed unknowingly via counterfeit Xanax® or Valium® preparations. As part of our Coronial workload, we observed an increase in the detection of bromazolam from September 2021 to November 2022. We report a series of 96 cases in which bromazolam was quantitated by high resolution accurate mass - mass spectrometry (HRAM - MS) in post-mortem blood. The mean (SD) post-mortem blood bromazolam concentration from our case series was 64.6 ( ± 79.4) µg/L (range <1-425 µg/L). Routine toxicological screening results have also been reported; the most commonly encountered drugs taken in combination with bromazolam were cocaine, gabapentinoids and diazepam. In 48% of cases at least one further designer benzodiazepine drug was also present (etizolam, flualprazolam, flubromazolam, flubromazepam). It is essential that laboratories providing toxicological investigations are aware of the limitations of their assays; and inclusion of bromazolam within targeted screening panels using LC-MS/MS is encouraged. Bromazolam has not been associated with death in isolation from resulting toxic concentrations; however, it is likely to enhance adverse clinical effects when taken in combination with stimulant and/or centrally-acting depressant drugs (poly-drug deaths). Bromazolam, similar to other benzodiazepines, may also impair cognition and decision making skills.
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Standardized clinical annotation of digital histopathology slides at the point of diagnosisAs digital pathology replaces conventional glass slide microscopy as a means of reporting cellular pathology samples, the annotation of digital pathology whole slide images is rapidly becoming part of a pathologist's regular practice. Currently, there is no recognizable organization of these annotations, and as a result, pathologists adopt an arbitrary approach to defining regions of interest, leading to irregularity and inconsistency and limiting the downstream efficient use of this valuable effort. In this study, we propose a Standardized Annotation Reporting Style for digital whole slide images. We formed a list of 167 commonly annotated entities (under 12 specialty subcategories) based on review of Royal College of Pathologists and College of American Pathologists documents, feedback from reporting pathologists in our NHS department, and experience in developing annotation dictionaries for PathLAKE research projects. Each entity was assigned a suitable annotation shape, SNOMED CT (SNOMED International) code, and unique color. Additionally, as an example of how the approach could be expanded to specific tumor types, all lung tumors in the fifth World Health Organization of thoracic tumors 2021 were included. The proposed standardization of annotations increases their utility, making them identifiable at low power and searchable across and between cases. This would aid pathologists reporting and reviewing cases and enable annotations to be used for research. This structured approach could serve as the basis for an industry standard and be easily adopted to ensure maximum functionality and efficiency in the use of annotations made during routine clinical examination of digital slides.
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Impact of changing from autopsy to post-mortem CT in an entire HM Coroner region due to a shortage of available pathologistsA significant problem facing routine medicolegal coroner-referred autopsies is a shortfall of pathologists prepared to perform them. This was particularly acute in Lancashire, where the coroner decided to initiate a service that relied on post-mortem computed tomography (PMCT). This involved training anatomical pathology technologists (APTs) to perform external examinations, radiographers to perform scans, and radiologists to interpret them. The service started in 2018 and now examines over 1,500 cases per year. This study outlines the PMCT process using NHS staff, with CT equipment and logistics managed by the commercial sector. It compares the demographics and outcomes of PM investigations for two 6-month periods: the autopsy service prior to 2018, and then the PMCT service. These data were then compared with previous UK PMCT data. Referrals for adult non-suspicious deaths were made in 913 cases of which 793 (87%) had PMCT between 01/10/2018 and 31/03/2019. Fifty-six cases had autopsy after PMCT, so 81% of cases potentially avoided autopsy. The PMCT service did not delay release of bodies to the next-of-kin. Comparing the cause of death given shows no difference in the proportions of natural and unnatural deaths. There was an increase in diagnosis of coronary artery disease for PMCT, with less respiratory diagnoses, a feature not previously demonstrated. These data suggest PMCT is a practical solution for potentially failing autopsy services. By necessity, this involves changes in diagnoses, as PMCT and autopsy have different strengths and weakness, but the ability to pick up unnatural death appears unaffected.
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Impact of the COVID-19 pandemic on a post-mortem CT service for adult non-suspicious deathDue to the COVID-19 pandemic, the post-mortem computed tomography (PMCT) service was expanded from three to seven cases per day to help mortuary services and avoid invasive autopsy. Additional targeted angiography and pulmonary ventilation procedures were stopped and triage rules relaxed to allow more indications to be scanned, including those requiring toxicology. A service evaluation was performed for the first 3-months of the COVID-19 pandemic compared to the equivalent period the previous year to study the impact of these changes. It was found that, despite the increase in deaths regionally, coronial referrals remained about 100 per month, a reduction in referral rate. The number undergoing PMCT rose from 28% to 74% of cases. Turnaround time remained the same. For cases triaged to PMCT, the need for subsequent autopsy increased from 7.9% to 15.8%. No significant changes were seen in diagnosis rates, including cardiac or respiratory. There was an increase in patients with coronary death without severe coronary calcification who underwent autopsy after PMCT. These may have been diagnosed by targeted coronary angiography. Fifty-three cases requiring toxicology/biochemistry had PMCT, with 38 having PMCT only. In 8/11 (72.7%) cases with normal PMCT and toxicology as the key diagnostic test, autopsy was performed prior to results. This suggests the pathology team were reluctant to risk an "unascertained" outcome. This study shows that it is possible to increase PMCT services by widening referral criteria and by limiting the use of enhanced imaging techniques, without significantly changing diagnosis rates of key diseases; however, selectively restarting targeted angiography may help avoid autopsy in some cases.
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Development and validation of artificial intelligence-based prescreening of large-bowel biopsies taken in the UK and Portugal: a retrospective cohort studyBACKGROUND: Histopathological examination is a crucial step in the diagnosis and treatment of many major diseases. Aiming to facilitate diagnostic decision making and improve the workload of pathologists, we developed an artificial intelligence (AI)-based prescreening tool that analyses whole-slide images (WSIs) of large-bowel biopsies to identify typical, non-neoplastic, and neoplastic biopsies. METHODS: This retrospective cohort study was conducted with an internal development cohort of slides acquired from a hospital in the UK and three external validation cohorts of WSIs acquired from two hospitals in the UK and one clinical laboratory in Portugal. To learn the differential histological patterns from digitised WSIs of large-bowel biopsy slides, our proposed weakly supervised deep-learning model (Colorectal AI Model for Abnormality Detection [CAIMAN]) used slide-level diagnostic labels and no detailed cell or region-level annotations. The method was developed with an internal development cohort of 5054 biopsy slides from 2080 patients that were labelled with corresponding diagnostic categories assigned by pathologists. The three external validation cohorts, with a total of 1536 slides, were used for independent validation of CAIMAN. Each WSI was classified into one of three classes (ie, typical, atypical non-neoplastic, and atypical neoplastic). Prediction scores of image tiles were aggregated into three prediction scores for the whole slide, one for its likelihood of being typical, one for its likelihood of being non-neoplastic, and one for its likelihood of being neoplastic. The assessment of the external validation cohorts was conducted by the trained and frozen CAIMAN model. To evaluate model performance, we calculated area under the convex hull of the receiver operating characteristic curve (AUROC), area under the precision-recall curve, and specificity compared with our previously published iterative draw and rank sampling (IDaRS) algorithm. We also generated heat maps and saliency maps to analyse and visualise the relationship between the WSI diagnostic labels and spatial features of the tissue microenvironment. The main outcome of this study was the ability of CAIMAN to accurately identify typical and atypical WSIs of colon biopsies, which could potentially facilitate automatic removing of typical biopsies from the diagnostic workload in clinics. FINDINGS: A randomly selected subset of all large bowel biopsies was obtained between Jan 1, 2012, and Dec 31, 2017. The AI training, validation, and assessments were done between Jan 1, 2021, and Sept 30, 2022. WSIs with diagnostic labels were collected between Jan 1 and Sept 30, 2022. Our analysis showed no statistically significant differences across prediction scores from CAIMAN for typical and atypical classes based on anatomical sites of the biopsy. At 0·99 sensitivity, CAIMAN (specificity 0·5592) was more accurate than an IDaRS-based weakly supervised WSI-classification pipeline (0·4629) in identifying typical and atypical biopsies on cross-validation in the internal development cohort (p<0·0001). At 0·99 sensitivity, CAIMAN was also more accurate than IDaRS for two external validation cohorts (p<0·0001), but not for a third external validation cohort (p=0·10). CAIMAN provided higher specificity than IDaRS at some high-sensitivity thresholds (0·7763 vs 0·6222 for 0·95 sensitivity, 0·7126 vs 0·5407 for 0·97 sensitivity, and 0·5615 vs 0·3970 for 0·99 sensitivity on one of the external validation cohorts) and showed high classification performance in distinguishing between neoplastic biopsies (AUROC 0·9928, 95% CI 0·9927-0·9929), inflammatory biopsies (0·9658, 0·9655-0·9661), and atypical biopsies (0·9789, 0·9786-0·9792). On the three external validation cohorts, CAIMAN had AUROC values of 0·9431 (95% CI 0·9165-0·9697), 0·9576 (0·9568-0·9584), and 0·9636 (0·9615-0·9657) for the detection of atypical biopsies. Saliency maps supported the representation of disease heterogeneity in model predictions and its association with relevant histological features. INTERPRETATION: CAIMAN, with its high sensitivity in detecting atypical large-bowel biopsies, might be a promising improvement in clinical workflow efficiency and diagnostic decision making in prescreening of typical colorectal biopsies. FUNDING: The Pathology Image Data Lake for Analytics, Knowledge and Education Centre of Excellence; the UK Government's Industrial Strategy Challenge Fund; and Innovate UK on behalf of UK Research and Innovation.
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Identification and characterisation of a rare MTTP variant underlying hereditary non-alcoholic fatty liver diseaseBackground & aims: Non-alcoholic fatty liver disease (NAFLD) is a complex trait with an estimated prevalence of 25% globally. We aimed to identify the genetic variant underlying a four-generation family with progressive NAFLD leading to cirrhosis, decompensation, and development of hepatocellular carcinoma in the absence of common risk factors such as obesity and type 2 diabetes. Methods: Exome sequencing and genome comparisons were used to identify the likely causal variant. We extensively characterised the clinical phenotype and post-prandial metabolic responses of family members with the identified novel variant in comparison with healthy non-carriers and wild-type patients with NAFLD. Variant-expressing hepatocyte-like cells (HLCs) were derived from human-induced pluripotent stem cells generated from homozygous donor skin fibroblasts and restored to wild-type using CRISPR-Cas9. The phenotype was assessed using imaging, targeted RNA analysis, and molecular expression arrays. Results: We identified a rare causal variant c.1691T>C p.I564T (rs745447480) in MTTP, encoding microsomal triglyceride transfer protein (MTP), associated with progressive NAFLD, unrelated to metabolic syndrome and without characteristic features of abetalipoproteinaemia. HLCs derived from a homozygote donor had significantly lower MTP activity and lower lipoprotein ApoB secretion than wild-type cells, while having similar levels of MTP mRNA and protein. Cytoplasmic triglyceride accumulation in HLCs triggered endoplasmic reticulum stress, secretion of pro-inflammatory mediators, and production of reactive oxygen species. Conclusions: We have identified and characterised a rare causal variant in MTTP, and homozygosity for MTTP p.I564T is associated with progressive NAFLD without any other manifestations of abetalipoproteinaemia. Our findings provide insights into mechanisms driving progressive NAFLD. Impact and implications: A rare genetic variant in the gene MTTP has been identified as responsible for the development of severe non-alcoholic fatty liver disease in a four-generation family with no typical disease risk factors. A cell line culture created harbouring this variant gene was characterised to understand how this genetic variation leads to a defect in liver cells, which results in accumulation of fat and processes that promote disease. This is now a useful model for studying the disease pathways and to discover new ways to treat common types of fatty liver disease.
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Screening of normal endoscopic large bowel biopsies with interpretable graph learning: a retrospective studyObjective: To develop an interpretable artificial intelligence algorithm to rule out normal large bowel endoscopic biopsies, saving pathologist resources and helping with early diagnosis. Design: A graph neural network was developed incorporating pathologist domain knowledge to classify 6591 whole-slides images (WSIs) of endoscopic large bowel biopsies from 3291 patients (approximately 54% female, 46% male) as normal or abnormal (non-neoplastic and neoplastic) using clinically driven interpretable features. One UK National Health Service (NHS) site was used for model training and internal validation. External validation was conducted on data from two other NHS sites and one Portuguese site. Results: Model training and internal validation were performed on 5054 WSIs of 2080 patients resulting in an area under the curve-receiver operating characteristic (AUC-ROC) of 0.98 (SD=0.004) and AUC-precision-recall (PR) of 0.98 (SD=0.003). The performance of the model, named Interpretable Gland-Graphs using a Neural Aggregator (IGUANA), was consistent in testing over 1537 WSIs of 1211 patients from three independent external datasets with mean AUC-ROC=0.97 (SD=0.007) and AUC-PR=0.97 (SD=0.005). At a high sensitivity threshold of 99%, the proposed model can reduce the number of normal slides to be reviewed by a pathologist by approximately 55%. IGUANA also provides an explainable output highlighting potential abnormalities in a WSI in the form of a heatmap as well as numerical values associating the model prediction with various histological features. Conclusion: The model achieved consistently high accuracy showing its potential in optimising increasingly scarce pathologist resources. Explainable predictions can guide pathologists in their diagnostic decision-making and help boost their confidence in the algorithm, paving the way for its future clinical adoption.
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Airborne transmission of respiratory viruses including severe acute respiratory syndrome coronavirus 2Purpose of review: The coronavirus disease 2019 pandemic has had a wide-ranging and profound impact on how we think about the transmission of respiratory viruses This review outlines the basis on which we should consider all respiratory viruses as aerosol-transmissible infections, in order to improve our control of these pathogens in both healthcare and community settings. Recent findings: We present recent studies to support the aerosol transmission of severe acute respiratory syndrome coronavirus 2, and some older studies to demonstrate the aerosol transmissibility of other, more familiar seasonal respiratory viruses. Summary: Current knowledge on how these respiratory viruses are transmitted, and the way we control their spread, is changing. We need to embrace these changes to improve the care of patients in hospitals and care homes including others who are vulnerable to severe disease in community settings.
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Sodium nitrite poisoning: A series of 20 fatalities in which post-mortem blood nitrite and nitrate concentrations are reportedSodium nitrite has several industrial applications however its accidental or intentional ingestion has been associated with severe toxicity and death. We present a series of 20 cases over 2 years in which evidence of sodium nitrite ingestion was found at the scene and supported by biochemical analysis of post-mortem blood nitrite and nitrate levels. Routine toxicological screening was performed on post-mortem blood samples received at University Hospitals of Leicester (UHL) NHS Trust, including ethanol analysis by headspace gas chromatography-flame ionisation detection (HS GC-FID), drug screening by high resolution accurate mass-mass spectrometry (HRAM-MS) and confirmatory drug quantitation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cases in which the history indicated the possibility of nitrite salts present at the scene, purchase of a suicide kit or a dusky-ash appearance of skin on post-mortem were referred to a specialist laboratory for nitrite and nitrate analysis. Analysis was based upon the gas-phase chemiluminescent reaction between nitric oxide (NO) and ozone; NO levels were determined using an NOA 280A, Sievers NO analyser. Twenty post-mortem cases in which sodium nitrite ingestion was the most probable cause of death were reported between January 2020 and February 2022; mean age was 31 years (range 14-49) with 9/20 (45%) female. 16/20 (80%) of cases had a history of depression and / or mental health issues. In half of the cases, anti-depressant / anti-psychotic drugs were prescribed; these drugs were detected in 8/20 (40%) cases. Ethanol was detected in 4/20 (20%) cases and anti-emetic drugs in 7/20 (35%) cases; anti-emetic drugs may be used to aid retention of sodium nitrite. Illicit drugs (amphetamine, cannabis and cocaine) were present in 3/20 cases (15%). Nitrite was found to be elevated in all but one case (95%), and nitrate was elevated in 17/20 (85%) cases. This paper highlights a surge in numbers of deaths across England and Wales due to sodium nitrite toxicity. Although, nitrite poisoning remains a rare cause of death, it is worthwhile considering its use in individuals with suicidal ideation given its unregulated availability online. The detection and quantitation of nitrite and nitrate requires specialised, highly reliable methodology currently only available in research laboratories. Implication of sodium nitrite ingestion also relies heavily upon circumstantial evidence combined with quantification. The provision of a quantitative nitrite / nitrate analytical service greatly assists in determining the cause of death in these cases.