Report of the 2022 Banff Heart Concurrent: Focus on non-human leukocyte antigen antibodies in rejection and the pathology of "mixed" rejection.

The Banff Heart Concurrent Session, held as part of the 16th Banff Foundation for Allograft Pathology Conference at Banff, Alberta, Canada, on September 21, 2022, focused on 2 major topics: non-human leukocyte antigen (HLA) antibodies and mixed rejection. Each topic was addressed in a multidisciplinary fashion with clinical, immunological, and pathology perspectives and future developments and prospectives. Following the Banff organization model and principles, the collective aim of the speakers on each topic was to • Determine current knowledge gaps in heart transplant pathology • Identify limitations of current pathology classification systems • Discuss next steps in addressing gaps and refining classification system.

The Banff 2022 Kidney Meeting Work Plan: Data-driven refinement of the Banff Classification for renal allografts.

The XVIth Banff Meeting for Allograft Pathology was held in Banff, Alberta, Canada, from September 19 to 23, 2022, as a joint meeting with the Canadian Society of Transplantation. In addition to a key focus on the impact of microvascular inflammation and biopsy-based transcript analysis on the Banff Classification, further sessions were devoted to other aspects of kidney transplant pathology, in particular T cell-mediated rejection, activity and chronicity indices, digital pathology, xenotransplantation, clinical trials, and surrogate endpoints. Although the output of these sessions has not led to any changes in the classification, the key role of Banff Working Groups in phrasing unanswered questions, and coordinating and disseminating results of investigations addressing these unanswered questions was emphasized. This paper summarizes the key Banff Meeting 2022 sessions not covered in the Banff Kidney Meeting 2022 Report paper and also provides an update on other Banff Working Group activities relevant to kidney allografts.

Discarded livers tested by normothermic machine perfusion in the VITTAL trial: Secondary end points and 5-year outcomes.

Normothermic machine perfusion (NMP) enables pretransplant assessment of high-risk donor livers. The VITTAL trial demonstrated that 71% of the currently discarded organs could be transplanted with 100% 90-day patient and graft survivals. Here, we report secondary end points and 5-year outcomes of this prospective, open-label, phase 2 adaptive single-arm study. The patient and graft survivals at 60 months were 82% and 72%, respectively. Four patients lost their graft due to nonanastomotic biliary strictures, one caused by hepatic artery thrombosis in a liver donated following brain death, and 3 in elderly livers donated after circulatory death (DCD), which all clinically manifested within 6 months after transplantation. There were no late graft losses for other reasons. All the 4 patients who died during the study follow-up had functioning grafts. Nonanastomotic biliary strictures developed in donated after circulatory death livers that failed to produce bile with pH >7.65 and bicarbonate levels >25 mmol/L. Histological assessment in these livers revealed high bile duct injury scores characterized by arterial medial necrosis. The quality of life at 6 months significantly improved in all but 4 patients suffering from nonanastomotic biliary strictures. This first report of long-term outcomes of high-risk livers assessed by normothermic machine perfusion demonstrated excellent 5-year survival without adverse effects in all organs functioning beyond 1 year (ClinicalTrials.gov number NCT02740608).

Digital pathology for reporting histopathology samples, including cancer screening samples - definitive evidence from a multisite study.

AIMS: 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.

Banff consensus recommendations for steatosis assessment in donor livers.

BACKGROUND AND AIMS: No consensus criteria or approaches exist regarding assessment of steatosis in the setting of human donor liver suitability for transplantation. The Banff Working Group on Liver Allograft Pathology undertook a study to determine the consistency with which steatosis is assessed and reported in frozen sections of potential donor livers. APPROACH AND RESULTS: A panel of 59 pathologists from 16 countries completed a questionnaire covering criteria used to assess steatosis in donor liver biopsies, including droplet size and magnification used; subsequently, steatosis severity was assessed in 18 whole slide images of donor liver frozen sections (n = 59). Survey results (from 56/59) indicated a wide variation in definitions and approaches used to assess and report steatosis. Whole slide image assessment led to a broad range in the scores. Findings were discussed at a workshop held at the 15th Banff Conference on Allograft Pathology, September 2019. The aims of discussions were to (i) establish consensus criteria for defining "large droplet fat" (LDF) that predisposes to increased risk of initial poor graft function and (ii) develop an algorithmic approach to determine fat droplet size and the percentage of hepatocytes involved. LDF was defined as typically a single fat droplet that expands the involved hepatocyte and is larger than adjacent nonsteatotic hepatocytes. Estimating severity of steatosis involves (i) low magnification estimate of the approximate surface area of the biopsy occupied by fat, (ii) higher magnification determination of the percentage of hepatocytes within the fatty area with LDF, and (iii) final score calculation. CONCLUSIONS: The proposed guidelines herein are intended to improve standardization in steatosis assessment of donor liver biopsies. The calculated percent LDF should be provided to the surgeon.

Introduction of the Concept of Diagnostic Sensitivity and Specificity of Normothermic Perfusion Protocols to Assess High-Risk Donor Livers.

Normothermic machine perfusion (NMP) allows objective assessment of donor liver transplantability. Several viability evaluation protocols have been established, consisting of parameters such as perfusate lactate clearance, pH, transaminase levels, and the production and composition of bile. The aims of this study were to assess 3 such protocols, namely, those introduced by the teams from Birmingham (BP), Cambridge (CP), and Groningen (GP), using a cohort of high-risk marginal livers that had initially been deemed unsuitable for transplantation and to introduce the concept of the viability assessment sensitivity and specificity. To demonstrate and quantify the diagnostic accuracy of these protocols, we used a composite outcome of organ use and 24-month graft survival as a surrogate endpoint. The effects of assessment modifications, including the removal of the most stringent components of the protocols, were also assessed. Of the 31 organs, 22 were transplanted after a period of NMP, of which 18 achieved the outcome of 24-month graft survival. The BP yielded 94% sensitivity and 50% specificity when predicting this outcome. The GP and CP both seemed overly conservative, with 1 and 0 organs, respectively, meeting these protocols. Modification of the GP and CP to exclude their most stringent components increased this to 11 and 8 organs, respectively, and resulted in moderate sensitivity (56% and 44%) but high specificity (92% and 100%, respectively) with respect to the composite outcome. This study shows that the normothermic assessment protocols can be useful in identifying potentially viable organs but that the balance of risk of underuse and overuse varies by protocol.

Myocardial fibrosis in asymptomatic and symptomatic chronic severe primary mitral regurgitation and relationship to tissue characterisation and left ventricular function on cardiovascular magnetic resonance.

BACKGROUND: Myocardial fibrosis occurs in end-stage heart failure secondary to mitral regurgitation (MR), but it is not known whether this is present before onset of symptoms or myocardial dysfunction. This study aimed to characterise myocardial fibrosis in chronic severe primary MR on histology, compare this to tissue characterisation on cardiovascular magnetic resonance (CMR) imaging, and investigate associations with symptoms, left ventricular (LV) function, and exercise capacity. METHODS: Patients with class I or IIa indications for surgery underwent CMR and cardiopulmonary exercise testing. LV biopsies were taken at surgery and the extent of fibrosis was quantified on histology using collagen volume fraction (CVFmean) compared to autopsy controls without cardiac pathology. RESULTS: 120 consecutive patients (64 ± 13 years; 71% male) were recruited; 105 patients underwent MV repair while 15 chose conservative management. LV biopsies were obtained in 86 patients (234 biopsy samples in total). MR patients had more fibrosis compared to 8 autopsy controls (median: 14.6% [interquartile range 7.4-20.3] vs. 3.3% [2.6-6.1], P < 0.001); this difference persisted in the asymptomatic patients (CVFmean 13.6% [6.3-18.8], P < 0.001), but severity of fibrosis was not significantly higher in NYHA II-III symptomatic MR (CVFmean 15.7% [9.9-23.1] (P = 0.083). Fibrosis was patchy across biopsy sites (intraclass correlation 0.23, 95% CI 0.08-0.39, P = 0.001). No significant relationships were identified between CVFmean and CMR tissue characterisation [native T1, extracellular volume (ECV) or late gadolinium enhancement] or measures of LV function [LV ejection fraction (LVEF), global longitudinal strain (GLS)]. Although the range of ECV was small (27.3 ± 3.2%), ECV correlated with multiple measures of LV function (LVEF: Rho = - 0.22, P = 0.029, GLS: Rho = 0.29, P = 0.003), as well as NTproBNP (Rho = 0.54, P < 0.001) and exercise capacity (%PredVO2max: R = - 0.22, P = 0.030). CONCLUSIONS: Patients with chronic primary MR have increased fibrosis before the onset of symptoms. Due to the patchy nature of fibrosis, CMR derived ECV may be a better marker of global myocardial status. Clinical trial registration Mitral FINDER study; Clinical Trials NCT02355418, Registered 4 February 2015, https://clinicaltrials.gov/ct2/show/NCT02355418.

Combined Hypothermic and Normothermic Machine Perfusion Improves Functional Recovery of Extended Criteria Donor Livers.

Hypothermic oxygenated perfusion (HOPE) and normothermic perfusion are seen as distinct techniques of ex situ machine perfusion of the liver. We aimed to demonstrate the feasibility of combining both techniques and whether it would improve functional parameters of donor livers into transplant standards. Ten discarded human donor livers had either 6 hours of normothermic perfusion (n = 5) or 2 hours of HOPE followed by 4 hours of normothermic perfusion (n = 5). Liver function was assessed according to our viability criteria; markers of tissue injury and hepatic metabolic activity were compared between groups. Donor characteristics were comparable. During the hypothermic perfusion phase, livers down-regulated mitochondrial respiration (oxygen uptake, P = 0.04; partial pressure of carbon dioxide perfusate, P = 0.04) and increased adenosine triphosphate levels 1.8-fold. Following normothermic perfusion, those organs achieved lower tissue expression of markers of oxidative injury (4-hydroxynonenal, P = 0.008; CD14 expression, P = 0.008) and inflammation (CD11b, P = 0.02; vascular cell adhesion molecule 1, P = 0.05) compared with livers that had normothermic perfusion alone. All livers in the combined group achieved viability criteria, whereas 40% (2/5) in the normothermic group failed (P = 0.22). In conclusion, this study suggests that a combined protocol of hypothermic oxygenated and normothermic perfusions might attenuate oxidative stress, tissue inflammation, and improve metabolic recovery of the highest-risk donor livers compared with normothermic perfusion alone.

Development of Clinical Criteria for Functional Assessment to Predict Primary Nonfunction of High-Risk Livers Using Normothermic Machine Perfusion.

Increased use of high-risk allografts is critical to meet the demand for liver transplantation. We aimed to identify criteria predicting viability of organs, currently declined for clinical transplantation, using functional assessment during normothermic machine perfusion (NMP). Twelve discarded human livers were subjected to NMP following static cold storage. Livers were perfused with a packed red cell-based fluid at 37°C for 6 hours. Multilevel statistical models for repeated measures were employed to investigate the trend of perfusate blood gas profiles and vascular flow characteristics over time and the effect of lactate-clearing (LC) and non-lactate-clearing (non-LC) ability of the livers. The relationship of lactate clearance capability with bile production and histological and molecular findings were also examined. After 2 hours of perfusion, median lactate concentrations were 3.0 and 14.6 mmol/L in the LC and non-LC groups, respectively. LC livers produced more bile and maintained a stable perfusate pH and vascular flow >150 and 500 mL/minute through the hepatic artery and portal vein, respectively. Histology revealed discrepancies between subjectively discarded livers compared with objective findings. There were minimal morphological changes in the LC group, whereas non-LC livers often showed hepatocellular injury and reduced glycogen deposition. Adenosine triphosphate levels in the LC group increased compared with the non-LC livers. We propose composite viability criteria consisting of lactate clearance, pH maintenance, bile production, vascular flow patterns, and liver macroscopic appearance. These have been tested successfully in clinical transplantation. In conclusion, NMP allows an objective assessment of liver function that may reduce the risk and permit use of currently unused high-risk livers.

Improved access to histopathology using a digital system could increase the organ donor pool and improve allocation.

Improvements in digital slide scanners have reached a stage that digital whole slide images (WSIs) can be used for diagnostic purposes. A digital system for histopathology, analogous to the systems used in radiology, would allow the establishment of networks of subspecialist histopathologists to provide a regional, national or even international rota to support out of hours histopathology for emergency frozen sections, urgent paraffin sections and to generally improve efficiencies with the provision of histopathology services. Such a system would promote appropriate organ utilization by allowing rapid characterization of unexpected lesions in the donor to determine whether donation should occur and further characterization of the organ, such as the degree of fibrosis in the kidney or steatosis in the liver, to determine whether the organ should be used. If introduced across Europe, this would promote safe and effective exchange of organs and support a cost efficient use of pathologist expertise. This review article outlines current issues with the provision of an urgent out of hours histopathology service and focuses on how such a service has the potential to increase organ donors, improve allocation, sharing and the use of available donor organs.

What factors influence cellular pathologists' confidence in case reporting?

Histopathology is a challenging interpretive discipline, and the level of confidence a pathologist has in their diagnosis is known to vary, which is conveyed descriptively in pathology reports. There has been little study to accurately quantify pathologists' diagnostic confidence or the factors that influence it. In this study involving sixteen pathologists from six NHS trusts, we assessed diagnostic confidence across multiple variables and four specialties. Each case was reported by four pathologists, with each pathologist reporting each case twice (on light microscopy (LM) and digital pathology (DP)). For each diagnosis, pathologists recorded their confidence on a 7-point Likert scale. This provided 16,187 diagnoses and associated confidence scores for analysis. All variables investigated were found to be significantly predictive of diagnostic confidence, except level of pathologist experience. Confidence was lower for difficult to report cases, cases where there was inter- and intra-pathologist variation in the diagnosis, and cases where the pathologist made an incorrect diagnosis. Confidence was higher, although nominally, for LM diagnoses than DP (rate ratio 1.09 (95% CI 1.01-1.18), p = 0.035), although results indicate pathologists are confident to report on DP. Lowest confidence scores were seen in areas of known diagnostic complexity and cases with quality issues. High confidence in incorrect diagnoses were almost invariably attributed to interpretive diagnostic differences which occurred across both rare and common lesions. The results highlight the value of external quality control schemes and the benefits of selective peer review when reporting.

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.

CD4+CD28- T cell expansion in granulomatosis with polyangiitis (Wegener's) is driven by latent cytomegalovirus infection and is associated with an increased risk of infection and mortality.

OBJECTIVE: Expanded populations of CD4+CD28- T cells with a cytotoxic phenotype have been repeatedly reported in patients with granulomatosis with polyangiitis (Wegener's) (GPA). In healthy individuals expansion of this T cell population follows cytomegalovirus (CMV) infection. We undertook this study to investigate whether CMV infection may be responsible for driving the expansion of CD4+CD28- T cells in GPA patients and how this might relate to clinical features. METHODS: Forty-eight GPA patients and 38 age-matched healthy donors were included in the study. CMV-specific IgG in serum was detected by enzyme-linked immunosorbent assay. Flow cytometric analysis was used to study T cell populations and phenotype. The presence of CMV in renal biopsy tissue from GPA patients was investigated by immunohistochemistry and polymerase chain reaction (PCR). Clinical information was obtained from patient records. RESULTS: Populations of CD4+CD28- T cells were only expanded in CMV-seropositive GPA patients and controls. In CMV-seropositive GPA patients we observed negative correlations between the percentages of CD4+CD28- T cells and both the percentage of naive T cells and the glomerular filtration rate at presentation. There was a significant association between the percentage of CD4+CD28- T cells and risk of infection and mortality. CMV could not be detected in renal tissue by PCR or immunohistochemistry. CMV seropositivity itself was not a risk factor for infection in a cohort of 182 patients with antineutrophil cytoplasmic antibody-associated vasculitis who had been recruited into clinical trials performed by the European Vasculitis Study Group. CONCLUSION: The expansion of CD4+CD28- T cells in GPA patients is associated with CMV infection and leads to a reduction in the number of naive T cells in peripheral blood. Patients with expanded CD4+CD28- T cells have significantly increased mortality and risk of infection.