Consensus recommendations for histological criteria of autoimmune hepatitis from the International AIH Pathology Group: Results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology: Results of a workshop on AIH histology hosted by the European Reference Network on Hepatological Diseases and the European Society of Pathology.

BACKGROUND & AIMS: Diagnostic histological criteria for autoimmune hepatitis (AIH) have not been clearly established. Previously published criteria focused mainly on chronic AIH, in which inflammatory changes mainly occur in portal/periportal regions and may not be applicable to acute presentation of AIH, in which inflammatory changes are typically predominantly lobular in location. International consensus criteria for the diagnosis and assessment of disease severity in both acute and chronic AIH are thus urgently needed. METHODS: Seventeen expert liver pathologists convened at an international workshop and subsequently used a modified Delphi panel approach to establish consensus criteria for the histopathological diagnosis of AIH. RESULTS: The consensus view is that liver biopsy should remain standard for diagnosing AIH. AIH is considered likely, if there is a predominantly portal lymphoplasmacytic hepatitis with more than mild interface activity and/or more than mild lobular hepatitis in the absence of histological features suggestive of another liver disease. AIH is also considered likely if there is predominantly lobular hepatitis with or without centrilobular necroinflammation and at least one of the following features: portal lymphoplasmacytic hepatitis, interface hepatitis or portal-based fibrosis, in the absence of histological features suggestive of another liver disease. Emperipolesis and hepatocellular rosettes are not regarded as being specific for AIH. CONCLUSIONS: The criteria proposed in this consensus statement provide a uniform approach to the histological diagnosis of AIH, which is relevant for patients with an acute as well as a chronic presentation and to more accurately reflect the current understanding of liver pathology in AIH.

STAT3-mediated upregulation of the AIM2 DNA sensor links innate immunity with cell migration to promote epithelial tumourigenesis.

OBJECTIVE: The absent in melanoma 2 (AIM2) cytosolic pattern recognition receptor and DNA sensor promotes the pathogenesis of autoimmune and chronic inflammatory diseases via caspase-1-containing inflammasome complexes. However, the role of AIM2 in cancer is ill-defined. DESIGN: The expression of AIM2 and its clinical significance was assessed in human gastric cancer (GC) patient cohorts. Genetic or therapeutic manipulation of AIM2 expression and activity was performed in the genetically engineered gp130 F/F spontaneous GC mouse model, as well as human GC cell line xenografts. The biological role and mechanism of action of AIM2 in gastric tumourigenesis, including its involvement in inflammasome activity and functional interaction with microtubule-associated end-binding protein 1 (EB1), was determined in vitro and in vivo. RESULTS: AIM2 expression is upregulated by interleukin-11 cytokine-mediated activation of the oncogenic latent transcription factor STAT3 in the tumour epithelium of GC mouse models and patients with GC. Genetic and therapeutic targeting of AIM2 in gp130 F/F mice suppressed tumourigenesis. Conversely, AIM2 overexpression augmented the tumour load of human GC cell line xenografts. The protumourigenic function of AIM2 was independent of inflammasome activity and inflammation. Rather, in vivo and in vitro AIM2 physically interacted with EB1 to promote epithelial cell migration and tumourigenesis. Furthermore, upregulated expression of AIM2 and EB1 in the tumour epithelium of patients with GC was independently associated with poor patient survival. CONCLUSION: AIM2 can play a driver role in epithelial carcinogenesis by linking cytokine-STAT3 signalling, innate immunity and epithelial cell migration, independent of inflammasome activation.

Collagen and elastic fibres in acute and chronic liver injury.

The histological distinction between acute and chronic liver injury is a challenging aspect of liver histopathology. It is traditionally based on the interpretation of morphological changes to the extracellular matrix (ECM) at sites of hepatocyte loss using histochemical stains. Our aim was to investigate whether immunohistochemistry and multiplexing for collagen type (I & III) and elastic fibres and a modified Victoria blue method could be helpful. We studied 43 livers removed at transplantation for acute liver failure (ALF, 20 cases) or cirrhosis (23) plus 8 normal controls. In ALF the periportal ECM was normal in 2 cases, contained mainly collagen I associated with a ductular reaction in 6 cases, and delicate elastic strands in 11 cases. Periportal deposition of mainly collagen I and mature elastic fibres was observed in cirrhosis. In ALF the perisinusoidal ECM was intact in 4 cases, collapsed or condensed but of normal composition (predominantly collagen III) in 2 cases, and collapsed and condensed containing mostly collagen I in 17 cases (7 including delicate immature elastic strands). In contrast, bridging fibrous septa of cirrhosis contained abundant collagen 1 and bundles of mature elastin. We propose the use of a scale and the use of immunohistochemistry and multiplexing in additional to histochemical stains to characterise the ECM changes in acute and chronic liver injury.

Constitutive STAT3 Serine Phosphorylation Promotes Helicobacter-Mediated Gastric Disease.

Gastric cancer is associated with chronic inflammation (gastritis) triggered by persistent Helicobacter pylori (H. pylori) infection. Elevated tyrosine phosphorylation of the latent transcription factor STAT3 is a feature of gastric cancer, including H. pylori-infected tissues, and aligns with nuclear transcriptional activity. However, the transcriptional role of STAT3 serine phosphorylation, which promotes STAT3-driven mitochondrial activities, is unclear. Here, by coupling serine-phosphorylated (pS)-STAT3-deficient Stat3SA/SA mice with chronic H. felis infection, which mimics human H. pylori infection in mice, we reveal a key role for pS-STAT3 in promoting Helicobacter-induced gastric pathology. Immunohistochemical staining for infiltrating immune cells and expression analyses of inflammatory genes revealed that gastritis was markedly suppressed in infected Stat3SA/SA mice compared with wild-type mice. Stomach weight and gastric mucosal thickness were also reduced in infected Stat3SA/SA mice, which was associated with reduced proliferative potential of infected Stat3SA/SA gastric mucosa. The suppressed H. felis-induced gastric phenotype of Stat3SA/SA mice was phenocopied upon genetic ablation of signaling by the cytokine IL-11, which promotes gastric tumorigenesis via STAT3. pS-STAT3 dependency by Helicobacter coincided with transcriptional activity on STAT3-regulated genes, rather than mitochondrial and metabolic genes. In the gastric mucosa of mice and patients with gastritis, pS-STAT3 was constitutively expressed irrespective of Helicobacter infection. Collectively, these findings suggest an obligate requirement for IL-11 signaling via constitutive pS-STAT3 in Helicobacter-induced gastric carcinogenesis.

Innate Immune Molecule NLRC5 Protects Mice From Helicobacter-induced Formation of Gastric Lymphoid Tissue.

BACKGROUND & AIMS: Helicobacter pylori induces strong inflammatory responses that are directed at clearing the infection, but if not controlled, these responses can be harmful to the host. We investigated the immune-regulatory effects of the innate immune molecule, nucleotide-binding oligomerization domain-like receptors (NLR) family CARD domain-containing 5 (NLRC5), in patients and mice with Helicobacter infection. METHODS: We obtained gastric biopsies from 30 patients in Australia. We performed studies with mice that lack NLRC5 in the myeloid linage (Nlrc5møKO) and mice without Nlrc5 gene disruption (controls). Some mice were gavaged with H pylori SS1 or Helicobacter felis; 3 months later, stomachs, spleens, and sera were collected, along with macrophages derived from bone marrow. Human and mouse gastric tissues and mouse macrophages were analyzed by histology, immunohistochemistry, immunoblots, and quantitative polymerase chain reaction. THP-1 cells (human macrophages, controls) and NLRC5-/- THP-1 cells (generated by CRISPR-Cas9 gene editing) were incubated with Helicobacter and gene expression and production of cytokines were analyzed. RESULTS: Levels of NLRC5 messenger RNA were significantly increased in gastric tissues from patients with H pylori infection, compared with patients without infection (P < .01), and correlated with gastritis severity (P < .05). H pylori bacteria induced significantly higher levels of chemokine and cytokine production by NLRC5-/- THP-1 macrophages than by control THP-1 cells (P < .05). After 3 months of infection with H felis, Nlrc5mø-KO mice developed gastric hyperplasia (P < .0001), splenomegaly (P < .0001), and increased serum antibody titers (P < .01), whereas control mice did not. Nlrc5mø-KO mice with chronic H felis infection had increased numbers of gastric B-cell follicles expressing CD19 (P < .0001); these follicles had features of mucosa-associated lymphoid tissue lymphoma. We identified B-cell-activating factor as a protein that promoted B-cell hyperproliferation in Nlrc5mø-KO mice. CONCLUSIONS: NLRC5 is a negative regulator of gastric inflammation and mucosal lymphoid formation in response to Helicobacter infection. Aberrant NLRC5 signaling in macrophages can promote B-cell lymphomagenesis during chronic Helicobacter infection.

Serine-Phosphorylated STAT3 Promotes Tumorigenesis via Modulation of RNA Polymerase Transcriptional Activity.

Deregulated activation of the latent oncogenic transcription factor STAT3 in many human epithelial malignancies, including gastric cancer, has invariably been associated with its canonical tyrosine phosphorylation and enhanced transcriptional activity. By contrast, serine phosphorylation (pS) of STAT3 can augment its nuclear transcriptional activity and promote essential mitochondrial functions, yet the role of pS-STAT3 among epithelial cancers is ill-defined. Here, we reveal that genetic ablation of pS-STAT3 in the gp130 F/F spontaneous gastric cancer mouse model and human gastric cancer cell line xenografts abrogated tumor growth that coincided with reduced proliferative potential of the tumor epithelium. Microarray gene expression profiling demonstrated that the suppressed gastric tumorigenesis in pS-STAT3-deficient gp130 F/F mice associated with reduced transcriptional activity of STAT3-regulated gene networks implicated in cell proliferation and migration, inflammation, and angiogenesis, but not mitochondrial function or metabolism. Notably, the protumorigenic activity of pS-STAT3 aligned with its capacity to primarily augment RNA polymerase II-mediated transcriptional elongation, but not initiation, of STAT3 target genes. Furthermore, by using a combinatorial in vitro and in vivo proteomics approach based on the rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) assay, we identified RuvB-like AAA ATPase 1 (RUVBL1/Pontin) and enhancer of rudimentary homolog (ERH) as interacting partners of pS-STAT3 that are pivotal for its transcriptional activity on STAT3 target genes. Collectively, these findings uncover a hitherto unknown transcriptional role and obligate requirement for pS-STAT3 in gastric cancer that could be extrapolated to other STAT3-driven cancers. SIGNIFICANCE: These findings reveal a new transcriptional role and mandatory requirement for constitutive STAT3 serine phosphorylation in gastric cancer.

Targeting mTOR and Src restricts hepatocellular carcinoma growth in a novel murine liver cancer model.

Liver cancer is a poor prognosis cancer with limited treatment options. To develop a new therapeutic approach, we derived HCC cells from a known model of murine hepatocellular carcinoma (HCC). We treated adiponectin (APN) knock-out mice with the carcinogen diethylnitrosamine, and the resulting tumors were 7-fold larger than wild-type controls. Tumors were disassociated from both genotypes and their growth characteristics evaluated. A52 cells from APN KO mice had the most robust growth in vitro and in vivo, and presented with pathology similar to the parental tumor. All primary tumors and cell lines exhibited activity of the mammalian target of Rapamycin (mTOR) and Src pathways. Subsequent combinatorial treatment, with the mTOR inhibitor Rapamycin and the Src inhibitor Dasatinib reduced A52 HCC growth 29-fold in vivo. Through protein and histological analyzes we observed activation of these pathways in human HCC, suggesting that targeting both mTOR and Src may be a novel approach for the treatment of HCC.

Acute-on-chronic liver failure 2018: a need for (urgent) liver biopsy?

INTRODUCTION: 'Acute-on-Chronic-Liver Failure (ACLF)' entered hepatology practice by the end of the 20th century. Although we lack precise and universally agreed definitions, acute decompensation of chronic liver disease with jaundice and deranged clotting, multi-organ failure and high, short-term mortality are hallmarks of the syndrome. Timely recognition and and treatment, including urgent liver transplantation, may save the life of certain patients. The diagnosis and management are mostly based on clinical features, but some have suggested to incorporate histopathology (liver biopsy). This may add to the differentiation between acute and chronic disease, primary and concomitant etiologies, and identify prognostic determinants. Areas covered: A review of the literature on ACLF and the outcome of the discussions at a topical international meeting on specific histopathological aspects of diagnosis and prognosis of the syndrome. Expert commentary: There is a lack of standardized descriptions of histopathological features and there is limited prospective experience with the role of pathology of ACLF. It is important for the clinical hepatologist to understand the potential and limitations of (transjugular) liver biopsy in ACLF and for the pathologist to help address the clinical question and recognise the histopathological features that help to characterize ACLF, both in terms of diagnosis and prognosis.

Hyperactive gp130/STAT3-driven gastric tumourigenesis promotes submucosal tertiary lymphoid structure development.

Tertiary lymphoid structures (TLSs) display phenotypic and functional characteristics of secondary lymphoid organs, and often develop in tissues affected by chronic inflammation, as well as in certain inflammation-associated cancers where they are prognostic of improved patient survival. However, the mechanisms that govern the development of tumour-associated TLSs remain ill-defined. Here, we observed tumour-associated TLSs in a preclinical mouse model (gp130F/F ) of gastric cancer, where tumourigenesis is dependent on hyperactive STAT3 signalling through the common IL-6 family signalling receptor, gp130. Gastric tumourigenesis was associated with the development of B and T cell-rich submucosal lymphoid aggregates, containing CD21+ cellular networks and high endothelial venules. Temporally, TLS formation coincided with the development of gastric adenomas and induction of homeostatic chemokines including Cxcl13, Ccl19 and Ccl21. Reflecting the requirement of gp130-driven STAT3 signalling for gastric tumourigenesis, submucosal TLS development was also STAT3-dependent, but independent of the cytokine IL-17 which has been linked with lymphoid neogenesis in chronic inflammation and autoimmunity. Interestingly, upregulated lymphoid chemokine expression and TLS formation were also observed in a chronic gastritis model induced by Helicobacter felis infection. Tumour-associated TLSs were also observed in patients with intestinal-type gastric cancer, and a gene signature linked with TLS development in gp130F/F mice was associated with advanced clinical disease, but was not prognostic of patient survival. Collectively, our in vivo data reveal that hyperactive gp130-STAT3 signalling closely links gastric tumourigenesis with lymphoid neogenesis, and while a TLS gene signature was associated with advanced gastric cancer in patients, it did not indicate a favourable prognosis.

Adiponectin confers protection from acute colitis and restricts a B cell immune response.

Adiponectin demonstrates beneficial effects in various metabolic diseases, including diabetes, and in bowel cancer. Recent data also suggest a protective role in colitis. However, the precise molecular mechanisms by which adiponectin and its receptors modulate colitis and the nature of the adaptive immune response in murine models are yet to be elucidated. Adiponectin knock-out mice were orally administered dextran sulfate sodium for 7 days and were compared with wild-type mice. The severity of disease was analyzed histopathologically and through cytokine profiling. HCT116 colonic epithelial cells were employed to analyze the in vitro effects of adiponectin and AdipoR1 interactions in colonic injury following dextran sulfate sodium treatment. Adiponectin knock-out mice receiving dextran sulfate sodium exhibited severe colitis, had greater inflammatory cell infiltration, and an increased presence of activated B cells compared with controls. This was accompanied by an exaggerated proinflammatory cytokine profile and increased STAT3 signaling. Adiponectin knock-out mouse colons had markedly reduced proliferation and increased epithelial apoptosis and cellular stress. In vitro, adiponectin reduced apoptotic, anti-proliferative, and stress signals and restored STAT3 signaling. Following the abrogation of AdipoR1 in vitro, these protective effects of adiponectin were abolished. In summary, adiponectin maintains intestinal homeostasis and protects against murine colitis through interactions with its receptor AdipoR1 and by modulating adaptive immunity.

Effects of Bariatric Surgery on Liver Function Tests in Patients with Nonalcoholic Fatty Liver Disease.

OBJECTIVES: Nonalcoholic fatty liver disease (NAFLD) affects over 80% of obese patients and is fueled by the metabolic syndrome. Weight loss is strongly advocated as a central treatment for NAFLD and has been shown to induce histological improvement. We aimed to define the patterns of improvement in NAFLD with weight loss and determine target weight goals for NAFLD resolution. METHODS: A prospective study of 84 morbidly obese patients with NAFLD undergoing bariatric surgery was conducted. Intraoperative liver biopsies were taken. Monthly follow-up, including blood tests and measurements, was performed. We monitored improvements in NAFLD by monthly alanine aminotransferase (ALT) and gamma glutamyltransferase (GGT) levels over 1 year. RESULTS: There was rapid improvement in ALT, particularly in the first 6 months following surgery, with statistically significant reduction in ALT at 2 months (35 vs 27 IU/L, p < 0.001). In multivariate analysis, there were significantly increased odds of ALT normalization after a %TBWL of 10-15% (odds ratio 2.49, p = 0.005). The odds of resolution increased with increasing weight loss. Triglyceride levels (odds ratio 0.59, p = 0.021) and baseline NAFLD activity score (odds ratio 0.28, p < 0.001) were also significantly related to ALT normalization. Improvements in ALT occurred prior to metabolic improvement and well before traditional ideal weight goals were reached. CONCLUSION: Improvements in NAFLD occurred rapidly after bariatric surgery and were closely related to weight loss and metabolic factors. A 10-15% reduction in body weight is an appropriate target to achieve substantial improvement in ALT levels. TRIAL REGISTRATION NUMBER: Australian Clinical Trials Registry (ACTRN12610000049077).

Modified thresholds for fibrosis risk scores in nonalcoholic fatty liver disease are necessary in the obese.

BACKGROUND: Obesity and its related comorbidities are significant risk factors for nonalcoholic fatty liver disease (NAFLD). Liver fibrosis is the major determinant of long-term outcomes in NAFLD. A non-invasive tool that accurately identifies obese patients at elevated risk of liver fibrosis would be of significant value. Fibrosis risk scores in patients with NAFLD have been proposed but have not been validated in obese populations. We aimed to validate established simple fibrosis scores in bariatric surgical patients. METHODS: We conducted a prospective study of 107 consecutive high-risk obese patients undergoing primary bariatric surgery. Proposed fibrosis scores (NAFLD fibrosis score; body mass index (BMI), aspartate aminotransferase (AST)/alanine aminotransferase ratio (ALT), and diabetes (BARD); Fibrosis-4 (FIB-4); Forn; and AST to platelet ratio index) were calculated and compared hepatic fibrosis determined by histology of intraoperative liver biopsies. Accuracy was determined, and fibrosis score thresholds were optimized. These modified thresholds were then validated in an independent bariatric surgical population. RESULTS: Liver biopsies were available in 101 patients. Sixty-eight patients had some degree of fibrosis, with 23 patients (23 %) having significant fibrosis (F2-4). The Forn score best predicted significant fibrosis (area under the receiver operator characteristic curve (AUROC) 0.724, p = 0.001). With standard thresholds, the sensitivity for the Forn score for identification of significant fibrosis (F2-4) was 0 %. Using modified thresholds of 3.5, the sensitivity and negative predictive value increased to 85.7 and 94.7 %. This threshold was applied to an independent validation cohort with good accuracy. CONCLUSIONS: Fibrosis risk scores using simple markers have moderate success at delineating obese patients with significant NAFLD-related fibrosis. Thresholds, however, need to be lowered to maximize diagnostic accuracy in this cohort.

Stereological Analysis of Liver Biopsy Histology Sections as a Reference Standard for Validating Non-Invasive Liver Fat Fraction Measurements by MRI.

BACKGROUND AND AIMS: Validation of non-invasive methods of liver fat quantification requires a reference standard. However, using standard histopathology assessment of liver biopsies is problematical because of poor repeatability. We aimed to assess a stereological method of measuring volumetric liver fat fraction (VLFF) in liver biopsies and to use the method to validate a magnetic resonance imaging method for measurement of VLFF. METHODS: VLFFs were measured in 59 subjects (1) by three independent analysts using a stereological point counting technique combined with the Delesse principle on liver biopsy histological sections and (2) by three independent analysts using the HepaFat-Scan® technique on magnetic resonance images of the liver. Bland Altman statistics and intraclass correlation (IC) were used to assess the repeatability of each method and the bias between the methods of liver fat fraction measurement. RESULTS: Inter-analyst repeatability coefficients for the stereology and HepaFat-Scan® methods were 8.2 (95% CI 7.7-8.8)% and 2.4 (95% CI 2.2-2.5)% VLFF respectively. IC coefficients were 0.86 (95% CI 0.69-0.93) and 0.990 (95% CI 0.985-0.994) respectively. Small biases (≤3.4%) were observable between two pairs of analysts using stereology while no significant biases were observable between any of the three pairs of analysts using HepaFat-Scan®. A bias of 1.4±0.5% VLFF was observed between the HepaFat-Scan® method and the stereological method. CONCLUSIONS: Repeatability of the stereological method is superior to the previously reported performance of assessment of hepatic steatosis by histopathologists and is a suitable reference standard for validating non-invasive methods of measurement of VLFF.

Role of aetiology in the progression, regression, and parenchymal remodelling of liver disease: implications for liver biopsy interpretation.

Clinicopathological concepts on acute and chronic liver disease have evolved rapidly during the last few years, with advances in general and specific treatment options and improved patient outcomes. The old paradigm of 'irreversibility' of cirrhosis had been challenged in major ways, and the validity of the usage of the term 'cirrhosis' has come into question. This paper addresses aetiology-based clinicopathological concepts and features that may deserve attention because they may determine disease outcome and, specifically, patterns of regression and remodelling. A variety of therapeutic interventions may influence remaining disease features after elimination of damaging agents (virus, alcohol, etc.), and determine the final clinical outcome including the risk of hepatocellular carcinoma (HCC). New concepts create new responsibilities and opportunities for the pathologist to contribute to the understanding of liver pathology and communicate this with clinical colleagues and researchers.

Aceruloplasminaemia: a rare but important cause of iron overload.

We present a case of a 20-year-old man referred to our service with iron overload and mildly deranged liver biochemistry. Although liver histopathology was consistent with haemochromatosis, iron studies were not consistent with this diagnosis. Serum ceruloplasmin levels were undetectable, leading to a diagnosis of aceruloplasminaemia. Unlike other iron overload disorders, neurological complications are a unique feature of this illness, and often irreversible, once established. The patient was treated with iron chelation prior to the onset of neurological injury, and experienced progressive normalisation of his ferritin and liver biochemistry. This is one of the youngest diagnosed cases in the published literature and, crucially, was a rare case of diagnosis and treatment prior to the onset of neurological sequelae. This is presented alongside a review of previously published cases of aceruloplasminaemia, including responses to iron chelation therapy.

Interrelationships of serum androgens, omental adipose tissue metabolism, and nonalcoholic fatty liver disease in obese premenopausal women.

BACKGROUND: The concept that androgens regulate multiple aspects of adipose tissue metabolism in women is based on studies of women with pathological androgen excess and in vitro studies generally using supraphysiological androgen concentrations. We investigated whether in women with normal-range serum testosterone, relationships exist between serum androgens and expression of proinflammatory (tumor necrosis factor-α, interleukin-6, CD68), anti-inflammatory (adiponectin), and lipid metabolic (lipoprotein lipase, hormone sensitive lipase) genes in omental adipose tissue, and severity of nonalcoholic fatty liver disease (NAFLD). METHODS: We studied obese women undergoing laparoscopic gastric band surgery (premenopausal, regular menses, nondiabetic, serum testosterone <2.5 nmol/L, n=27). Gene expression was measured in omental adipose tissue. Liver biopsies were examined in 22 participants. RESULTS: Serum testosterone or androstanediol glucuronide (an indicator of peripheral androgen metabolism) were not related to markers of inflammation or lipid metabolism in omental adipose tissue. In women with NAFLD, there was a significant trend to higher serum free testosterone, serum insulin, and insulin resistance with increasing severity of liver pathology; however, markers of inflammation in omental adipose tissue did not differ. Omental lipoprotein lipase expression was significantly increased in women with nonalcoholic steatohepatitis (NASH). CONCLUSIONS: These primarily correlative data suggest that physiological-range androgen levels do not influence inflammation or lipid metabolism in omental adipose tissue of women, but further studies of direct androgen effects on adipose tissue are needed for confirmation. Androgens may still play a role in the pathogenesis of NAFLD in women, via mechanisms unrelated to omental adipose tissue metabolism.

Hepatic steatosis estimated microscopically versus digital image analysis.

BACKGROUND & AIMS: Evaluate in liver biopsies: (i) interobserver agreement of estimates of fat proportionate area (eFPA) and steatosis grading, (ii) the relationship between steatosis grades and measured fat proportionate area (mFPA, digital image analysis), (iii) the accuracy of eFPA, (iv) to present images to aid standardization and accuracy of eFPA. METHODS: Twenty-one liver biopsies were selected from the Royal Free Hospital (RFH) histopathology archive to represent the full range of histopathological steatosis severity. As many non-overlapping fields of parenchyma as possible were photographed at ×20 objective magnification from the biopsies (n = 651). A total of 15 sample images were selected to represent the range of steatosis seen. Twelve hepatopathologists from 11 sites worldwide independently evaluated the sample images for steatosis grade [normal (none)/mild/moderate/severe], and eFPA (% area of liver parenchyma occupied by fat). RESULTS: The hepatopathologists had good linear correlation between eFPA and mFPA for sample images (r = 0.924, P < .001) and excellent concordance (kappa = 0.91, P < 0.001). Interobserver concordance of steatosis grade showed 'substantial agreement' (kappa = 0.64). There was significant difference between eFPA and mFPA in the sample images for mild, moderate and severe steatosis (P = 0.024, P < 0.001, P < 0.001 respectively): the observers consistently over-estimated the eFPA. CONCLUSION: Hepatopathologists showed 'excellent' interobserver agreement in eFPA and 'substantial' agreement in assigning steatosis grade (precision was high). However, compared with mFPA, eFPA was inaccurate. eFPA systematically exceeds mFPA; generally the overestimation increases with severity of steatosis. Considering that non-invasive technologies for estimating liver fat utilize histopathology as reference, such assessments would benefit from quantitative validation of visually estimated microscopic liver fat percentages.