Banff 2022 Liver Group Meeting report: Monitoring long-term allograft health.

The Banff Working Group on Liver Allograft Pathology met in September 2022. Participants included hepatologists, surgeons, pathologists, immunologists, and histocompatibility specialists. Presentations and discussions focused on the evaluation of long-term allograft health, including noninvasive and tissue monitoring, immunosuppression optimization, and long-term structural changes. Potential revision of the rejection classification scheme to better accommodate and communicate late T cell-mediated rejection patterns and related structural changes, such as nodular regenerative hyperplasia, were discussed. Improved stratification of long-term maintenance immunosuppression to match the heterogeneity of patient settings will be central to improving long-term patient survival. Such personalized therapeutics are in turn contingent on a better understanding and monitoring of allograft status within a rational decision-making approach, likely to be facilitated in implementation with emerging decision-support tools. Proposed revisions to rejection classification emerging from the meeting include the incorporation of interface hepatitis and fibrosis staging. These will be opened to online testing, modified accordingly, and subject to consensus discussion leading up to the next Banff conference.

Survey of liver pathologists to assess attitudes towards digital pathology and artificial intelligence.

AIMS: A survey of members of the UK Liver Pathology Group (UKLPG) was conducted, comprising consultant histopathologists from across the UK who report liver specimens and participate in the UK National Liver Pathology External Quality Assurance scheme. The aim of this study was to understand attitudes and priorities of liver pathologists towards digital pathology and artificial intelligence (AI). METHODS: The survey was distributed to all full consultant members of the UKLPG via email. This comprised 50 questions, with 48 multiple choice questions and 2 free-text questions at the end, covering a range of topics and concepts pertaining to the use of digital pathology and AI in liver disease. RESULTS: Forty-two consultant histopathologists completed the survey, representing 36% of fully registered members of the UKLPG (42/116). Questions examining digital pathology showed respondents agreed with the utility of digital pathology for primary diagnosis 83% (34/41), second opinions 90% (37/41), research 85% (35/41) and training and education 95% (39/41). Fatty liver diseases were an area of demand for AI tools with 80% in agreement (33/41), followed by neoplastic liver diseases with 59% in agreement (24/41). Participants were concerned about AI development without pathologist involvement 73% (30/41), however, 63% (26/41) disagreed when asked whether AI would replace pathologists. CONCLUSIONS: This study outlines current interest, priorities for research and concerns around digital pathology and AI for liver pathologists. The majority of UK liver pathologists are in favour of the application of digital pathology and AI in clinical practice, research and education.

A case of membranous nephropathy with microspherular deposits progressing to collapsing nephropathy and dialysis dependency, but with full recovery following immunosuppression.

Membranous nephropathy with microspherular deposits is a rare renal condition associated with sub-nephrotic or nephrotic-range proteinuria. We report a case presenting with severe nephrotic syndrome and pathological features of collapsing glomerulopathy. This is the first case we are aware of that progressed to requiring dialysis. The patient received rituximab and corticosteroids. She has now been off dialysis for over a year with both serum creatinine and urine protein-creatinine ratio returning to baseline.

Utility of interval kidney biopsy in ANCA-associated vasculitis.

OBJECTIVES: ANCA-associated vasculitis (AAV) is a rare autoimmune disorder that commonly involves the kidney. Early identification of kidney involvement, assessing treatment-response and predicting outcome are important clinical challenges. Here, we assessed the potential utility of interval kidney biopsy in AAV. METHODS: In a tertiary referral centre with a dedicated vasculitis service, we identified patients with AAV who had undergone interval kidney biopsy, defined as a repeat kidney biopsy (following an initial biopsy showing active AAV) undertaken to determine the histological response in the kidney following induction immunosuppression. We analysed biochemical, histological and outcome data, including times to kidney failure and death for all patients. RESULTS: We identified 57 patients with AAV who underwent at least one interval kidney biopsy (59 interval biopsies in total; median time to interval biopsy ∼130 days). Of the 59 interval biopsies performed, 24 (41%) patients had clinically suspected active disease at time of biopsy which was confirmed histologically in only 42% of cases; 35 (59%) patients were in clinical disease-remission, and this was correct in 97% of cases. The clinician's impression was incorrect in one in four patients. Hematuria at interval biopsy did not correlate with histological activity. Interval biopsy showed fewer acute lesions and more chronic damage compared with initial biopsy and led to immunosuppressive treatment-change in 75% (44/59) of patients. Clinical risk prediction tools tended to operate better using interval biopsy data. CONCLUSION: Interval kidney biopsy is useful for determining treatment-response and subsequent disease management in AAV. It may provide better prognostic information than initial kidney biopsy and should be considered for inclusion into future clinical trials and treatment protocols for patients with AAV.

The use of digital magnification to reduce radiation dose in the cardiac catheter laboratory.

OBJECTIVES: To audit whether using magnification of images by use of a large viewing screen using digital matrix magnification which enlarges the image by 33% without using the X-ray machine zoom magnification protocols on a Siemens Artis Zee X-ray machine in a cardiac catheter laboratory results in a reduction of kerma-area product (KAP) for both diagnostic and interventional procedures. This reduction was predicted in an in vitro study in our laboratory, which has previously shown a 20.4% reduction in KAP. METHODS: A retrospective analysis was conducted of the radiation exposure to compare the measured KAP recorded during the period when conventional magnification with automatic brightness and dose control was used on a Siemens Artis Zee X-ray machine with a flat panel detector and when magnification settings were avoided by using a large screen to enlarge and project a non-magnified image by digital magnification. The analysis was carried out for patients having a diagnostic coronary angiogram and those having an interventional coronary procedure. RESULTS: For diagnostic coronary angiograms the median KAP per procedure in the period using conventional magnification was 2124.5 µGy.m2 compared to 1401 µGy.m2 when image matrix magnification was used, a 34% reduction (p < 0.0001). For interventional coronary procedures, the median KAP per procedure in the period using conventional magnification was 3791 µGy.m2 compared to 2568.5 µGy.m2 when image matrix magnification was used, a 32% reduction (p < 0.0001). CONCLUSION: Avoiding using conventional magnification in the cardiac catheter laboratory and using a large screen to magnify images was associated with a statistically significant greater than 30% reduction in KAP. ADVANCES IN KNOWLEDGE: This paper is the proof in clinical practice of a theoretical conclusion that radiation dose (KAP) is reduced by use of Image matrix magnification using a large viewing screen without the need to use X-ray tube magnification without significant loss of image resolution in interventional cardiology. The same approach will be useful in interventional radiology.

Tissue-Specific Immunopathology in Fatal COVID-19.

Rationale: In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown.Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury.Methods: Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by in situ viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence.Measurements and Main Results: Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues.Conclusions: Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.

Camk2n1 Is a Negative Regulator of Blood Pressure, Left Ventricular Mass, Insulin Sensitivity, and Promotes Adiposity.

Metabolic syndrome is a cause of coronary artery disease and type 2 diabetes mellitus. Camk2n1 resides in genomic loci for blood pressure, left ventricle mass, and type 2 diabetes mellitus, and in the spontaneously hypertensive rat model of metabolic syndrome, Camk2n1 expression is cis-regulated in left ventricle and fat and positively correlates with adiposity. Therefore, we knocked out Camk2n1 in spontaneously hypertensive rat to investigate its role in metabolic syndrome. Compared with spontaneously hypertensive rat, Camk2n1-/- rats had reduced cardiorenal CaMKII (Ca2+/calmodulin-dependent kinase II) activity, lower blood pressure, enhanced nitric oxide bioavailability, and reduced left ventricle mass associated with altered hypertrophic networks. Camk2n1 deficiency reduced insulin resistance, visceral fat, and adipogenic capacity through the altered cell cycle and complement pathways, independent of CaMKII. In human visceral fat, CAMK2N1 expression correlated with adiposity and genomic variants that increase CAMK2N1 expression associated with increased risk of coronary artery disease and type 2 diabetes mellitus. Camk2n1 regulates multiple networks that control metabolic syndrome traits and merits further investigation as a therapeutic target in humans.

Global quality assessment of liver allograft C4d staining during acute antibody-mediated rejection in formalin-fixed, paraffin-embedded tissue.

Discussion of liver antibody-mediated rejection during the 2011, 2013, and 2015 Banff liver sessions raised concerns over reliability of complement fragment 4d (C4d) staining, precipitating a global survey followed by a tissue microarray staining quality assessment study among centers on formalin-fixed, paraffin-embedded tissue. Tissue microarray sections containing tissue plugs of resected native and allograft (with acute antibody-mediated rejection) liver, heart, and kidney (n = 33 total cores) were sent to 31 centers for C4d staining using local method(s) and pathologist scoring. Digital whole-slide images (n = 40) were then semiquantitatively scored by 7 experts for background, distribution, and intensity of portal vein and capillary, hepatic artery, sinusoidal, and central vein endothelia and portal and central stromal staining. Results showed that strong and diffuse portal vein and capillary C4d staining, as determined by both local and central pathologists, clearly distinguished allografts showing acute antibody-mediated rejection from native livers and from those with evidence of weaker donor-specific antibody. Downstream vascular endothelial cell C4d staining and assessment were more variable and difficult to identify. C4d staining in the majority of laboratories reliably detects acute liver allograft antibody-mediated rejection in formalin-fixed, paraffin-embedded tissues. Assessment should focus on portal veins and capillaries, sinusoids, and central veins present in peripheral core needle biopsies. C4d staining in one organ does not always translate to staining in another.

Combined Papillary Renal Cell Carcinoma with Neuroendocrine Differentiation and Mucinous Tubular and Spindle Cell Carcinoma.

A unique case of combined papillary renal cell carcinoma (PRCC) and mucinous tubular and spindle cell carcinoma (MTSCC) presenting in a man aged 67 years is reported. The two separate components were distinct on morphological, immunohistochemical (IHC), and genetic grounds, while type 2 PRCC predominated. Three years after the initial diagnosis, the PRCC component metastasized to the lungs where it morphologically mimicked a pulmonary neuroendocrine tumor. Retrospectively focal neuroendocrine differentiation was demonstrated by IHC in the PRCC component of the primary neoplasm.

Detection of viral hepatitis E in clinical liver biopsies.

AIMS: To determine the relative utility of in-situ testing for hepatitis E virus (HEV) RNA and paraffin-section polymerase chain reaction (PCR) to diagnose HEV infection in paraffin-embedded clinical liver biopsies, and to correlate with clinicopathological characteristics. METHODS AND RESULTS: We evaluated in-situ and quantitative PCR (qPCR)-based approaches to identifying HEV in clinical liver biopsies from infected patients from multiple centres, correlating with clinical setting (immunocompetent, allograft or immunosuppressed native liver) and histological findings. Thirty-six biopsies from 29 patients had histological data, 27 and 23 of which had satisfactory material for in-situ RNA testing and tissue qPCR, respectively. Both approaches specifically identified HEV infection, but tissue qPCR was significantly more sensitive than RNAscope in-situ testing (P = 0.035). In immunocompetent but not immunosuppressed patients the tissue qPCR yield correlated with the severity of lobular hepatitis (rho = 0.94, P < 0.001). qPCR viral yield was comparably high in allografts and immunosuppressed native livers and significantly greater than with native liver infection. Immunosuppressed patients showed reduced severity of hepatitis and cholestatic changes, compared with immunocompetent patients. Indeed, HEV-infected liver allografts could show minimal hepatitis for many months. In individual cases each technique was useful when serum was not available to identify chronic infection retrospectively (in biopsies taken 4-31 months before diagnosis), to identify persistent/residual infection when contemporary serum PCR was negative and to identify cleared infection. CONCLUSIONS: qPCR is more effective than in-situ RNA testing to identify HEV infection in paraffin-embedded liver biopsies and has diagnostic utility in selected settings.

Bimodal dynamics of granular organelles in primary renin-expressing cells revealed using TIRF microscopy.

Renin is the initiator and rate-limiting factor in the renin-angiotensin blood pressure regulation system. Although renin is not exclusively produced in the kidney, in nonmurine species the synthesis and secretion of the active circulatory enzyme is confined almost exclusively to the dense core granules of juxtaglomerular (JG) cells, where prorenin is processed and stored for release via a regulated pathway. Despite its importance, the structural organization and regulation of granules within these cells is not well understood, in part due to the difficulty in culturing primary JG cells in vitro and the lack of appropriate cell lines. We have streamlined the isolation and culture of primary renin-expressing cells suitable for high-speed, high-resolution live imaging using a Percoll gradient-based procedure to purify cells from RenGFP+ transgenic mice. Fibronectin-coated glass coverslips proved optimal for the adhesion of renin-expressing cells and facilitated live cell imaging at the plasma membrane of primary renin cells using total internal reflection fluorescence microscopy (TIRFM). To obtain quantitative data on intracellular function, we stained mixed granule and lysosome populations with Lysotracker Red and stimulated cells using 100 nM isoproterenol. Analysis of membrane-proximal acidic granular organelle dynamics and behavior within renin-expressing cells revealed the existence of two populations of granular organelles with distinct functional responses following isoproterenol stimulation. The application of high-resolution techniques for imaging JG and other specialized kidney cells provides new opportunities for investigating renal cell biology.

Working with Commercially Available Quantum Dots for Immunofluorescence on Tissue Sections.

Quantum dots are semiconductor fluorescent nanocrystals that exhibit excellent characteristics compared with more commonly used organic fluorescent dyes. For many years quantum dot conjugated products have been available in multiple forms for fluorescence imaging of tissue sections under the trademark name Qdot®. They have much increased brightness, narrow emission spectrum, large Stokes shift and photostability compared with conventional organic fluorescent dyes, which together make them the fluorophores of choice for demanding requirements. Vivid Qdots are recent replacements for original Qdots, modified to improve brightness, however this has affected the fluorescence stability in commonly used conditions for immunohistochemistry. We present here our investigation of the stability of original and Vivid Qdots in solution and in immunohistochemistry, highlight the potential pitfalls and propose a protocol for stable and reliable multiplex staining with current commercially available original and Vivid Qdots.

Choice of Illumination System & Fluorophore for Multiplex Immunofluorescence on FFPE Tissue Sections.

The recent availability of novel dyes and alternative light sources to facilitate complex tissue immunofluorescence studies such as multiplex labelling has not been matched by reports critically evaluating the considerations and relative benefits of these new tools, particularly in combination. Product information is often limited to wavelengths used for older fluorophores (FITC, TRITC & corresponding Alexa dyes family). Consequently, novel agents such as Quantum dots are not widely appreciated or used, despite highly favourable properties including extremely bright emission, stability and potentially reduced tissue autofluorescence at the excitation wavelength. Using spectral analysis, we report here a detailed critical appraisal and comparative evaluation of different light sources and fluorophores in multiplex immunofluorescence of clinical biopsy sections. The comparison includes mercury light, metal halide and 3 different LED-based systems, using 7 Qdots (525, 565, 585, 605, 625, 705), Cy3 and Cy5. We discuss the considerations relevant to achieving the best combination of light source and fluorophore for accurate multiplex fluorescence quantitation. We highlight practical limitations and confounders to quantitation with filter-based approaches.

Hemin Preconditioning Upregulates Heme Oxygenase-1 in Deceased Donor Renal Transplant Recipients: A Randomized, Controlled, Phase IIB Trial.

BACKGROUND: The enzyme heme oxygenase-1 (HO-1) degrades heme and protects against ischemia-reperfusion injury. Monocytes/macrophages are the major source of HO-1 and higher levels improve renal transplant outcomes. Heme arginate (HA) safely induces HO-1 in humans. METHODS: The Heme Oxygenase-1 in renal Transplantation study was a randomized, placebo-controlled, IIb trial to evaluate HA effect on HO-1 upregulation after deceased donor kidney transplantation. 40 recipients were randomized to either 3 mg kg HA or placebo (0.9% NaCl), given preoperatively (day 0) and again on day 2. Recipient blood and urine were collected daily. Graft biopsies were taken preoperatively and on day 5. Primary outcome was HO-1 upregulation in peripheral blood mononuclear cells (PBMCs). Secondary outcomes were graft HO-1 upregulation and injury, urinary biomarkers, and renal function. RESULTS: The HA upregulated PBMC HO-1 protein more than placebo at 24 hours: HA 11.1 ng/mL versus placebo 0.14 ng/mL (P = < 0.0001). The PBMC HO-1 messenger RNA also increased: HA 2.73-fold versus placebo 1.41-fold (P = 0.02). Heme arginate increased day 5 tissue HO-1 protein immunopositivity compared with placebo: HA 0.21 versus placebo -0.03 (P = 0.02) and % HO-1-positive renal macrophage also increased: HA 50.8 cells per high power field versus placebo 22.3 (P = 0.012). Urinary biomarkers were reduced after HA but not significantly. Histological injury and renal function were similar but the study was not powered for this. Adverse events were equivalent between groups. CONCLUSIONS: The primary outcome was achieved and demonstrated for the first time that HA safely induces HO-1 in transplant recipients. Planned larger studies will determine the impact of HO-1 upregulation on clinical outcomes and evaluate the benefit to patients at risk of ischemia-reperfusion injury.

Following specific podocyte injury captopril protects against progressive long term renal damage.

BACKGROUND: Angiotensin converting enzyme inhibitors (ACEi) reduce proteinuria and preserve kidney function in proteinuric renal diseases. Their nephroprotective effect exceeds that attributable to lowering of blood pressure alone. This study examines the potential of ACEi to protect from progression of injury after a highly specific injury to podocytes in a mouse model. METHODS: We created transgenic (Podo-DTR) mice in which graded specific podocyte injury could be induced by a single injection of diphtheria toxin. Transgenic and wild-type mice were given the ACEi captopril in drinking water, or water alone, commencing 24h after toxin injection. Kidneys were examined histologically at 8 weeks and injury assessed by observers blinded to experimental group. RESULTS: After toxin injection, Podo-DTR mice developed acute proteinuria, and at higher doses transient renal impairment, which subsided within 3 weeks to be followed by a slow glomerular scarring process. Captopril treatment in Podo-DTR line 57 after toxin injection at 5ng/g body weight reduced proteinuria and ameliorated glomerular scarring, matrix accumulation and glomerulosclerosis almost to baseline (toxin: 17%; toxin + ACEi 10%, p<0.04; control 7% glomerular scarring). Podocyte counts were reduced after toxin treatment and showed no recovery irrespective of captopril treatment (7.1 and 7.3 podocytes per glomerular cross section in water and captopril-treated animals compared with 8.2 of wild-type controls, p<0.05). CONCLUSIONS: Observations in Podo-DTR mice support the hypothesis that continuing podocyte dysfunction is a key abnormality in proteinuric disease. Our model is ideal for studying strategies to protect the kidney from progressive injury following podocyte depletion. Demonstrable protective effects from captopril occur, despite indiscernible preservation or restoration of podocyte counts, at least after this degree of relatively mild injury.

Benefits of an expanded use of plasma exchange for anti-neutrophil cytoplasmic antibody-associated vasculitis within a dedicated clinical service.

BACKGROUND: Current recommendations for ANCA-associated vasculitis (AAV) support its management within a dedicated clinical service. Therapies for AAV are imperfect with many patients failing to achieve disease control and others experiencing disease relapse. Plasma exchange (PEX) may be beneficial especially when the kidney is involved. METHODS: Within a new, dedicated service we retrospectively assessed, over a 6-year period, the benefits of PEX in two patient cohorts, discriminated by PEX treatment alone. Patients received PEX alongside standard of care if they fulfilled any of the following criteria: 1. serum creatinine >500 µmol/l or dialysis-requiring renal failure, 2. alveolar haemorrhage, 3. renal biopsy showing ≥30 % focal and necrotising lesions ± cellular crescents. Outcome measures included disease remission and relapse, cumulative immunosuppression, and morbidity and mortality. RESULTS: Of 104 new patients, 58 patients received PEX at presentation, 46 did not. Cyclophosphamide and/or rituximab dosing was similar for both groups. Although patients receiving PEX had poorer renal function, a higher C-reactive protein and disease activity score at presentation disease remission rate was similar in both groups (no PEX vs. PEX: 96 % vs. 98 %). The PEX group entered remission quicker (no PEX vs. PEX: 3.9 ± 4.0 vs. 2.8 ± 1.3 months, p < 0.05), with a lower 3-month cumulative glucocorticoid dose (no PEX vs. PEX: 2.5 ± 0.4 vs. 2.3 ± 0.2 g, p < 0.001). Relapse was similar between groups but adverse events lower in the PEX group. CONCLUSIONS: PEX may be of benefit in AAV. Larger, longer randomised controlled trials are now needed.

Acute Liver Injury Is Independent of B Cells or Immunoglobulin M.

BACKGROUND & AIMS: Acute liver injury is a clinically important pathology and results in the release of Danger Associated Molecular Patterns, which initiate an immune response. Withdrawal of the injurious agent and curtailing any pathogenic secondary immune response may allow spontaneous resolution of injury. The role B cells and Immunoglobulin M (IgM) play in acute liver injury is largely unknown and it was proposed that B cells and/or IgM would play a significant role in its pathogenesis. METHODS: Tissue from 3 models of experimental liver injury (ischemia-reperfusion injury, concanavalin A hepatitis and paracetamol-induced liver injury) and patients transplanted following paracetamol overdose were stained for evidence of IgM deposition. Mice deficient in B cells (and IgM) were used to dissect out the role B cells and/or IgM played in the development or resolution of injury. Serum transfer into mice lacking IgM was used to establish the role IgM plays in injury. RESULTS: Significant deposition of IgM was seen in the explanted livers of patients transplanted following paracetamol overdose as well as in 3 experimental models of acute liver injury (ischemia-reperfusion injury, concanavalin A hepatitis and paracetamol-induced liver injury). Serum transfer into IgM-deficient mice failed to reconstitute injury (p = 0.66), despite successful engraftment of IgM. Mice deficient in both T and B cells (RAG1-/-) mice (p<0.001), but not B cell deficient (µMT) mice (p = 0.93), were significantly protected from injury. Further interrogation with T cell deficient (CD3εKO) mice confirmed that the T cell component is a key mediator of sterile liver injury. Mice deficient in B cells and IgM mice did not have a significant delay in resolution following acute liver injury. DISCUSSION: IgM deposition appears to be common feature of both human and murine sterile liver injury. However, neither IgM nor B cells, play a significant role in the development of or resolution from acute liver injury. T cells appear to be key mediators of injury. In conclusion, the therapeutic targeting of IgM or B cells (e.g. with Rituximab) would have limited benefit in protecting patients from acute liver injury.