Ashraf Mohammad El-Badry

Assessment of hepatic steatosis by expert pathologists: the end of a gold standard

Background:The presence of fat in the liver is considered a major risk for postoperative complication after liver surgery and transplantation. The current standard of quantification of hepatic steatosis is microscopic evaluation by pathologists, although consistency in such assessment remains unclear. Computerized image analysis is an alternative method for objective assessment of the degree of hepatic steatosis. Methods:High resolution images of hematoxylin and eosin stained liver sections from 46 consecutive patients, initially diagnosed with liver steatosis, were blindly assessed by 4 established expert pathologists from different institutions. Computerized analysis was carried out simultaneously on the same sections. Interobserver agreement and correlation between the pathologists’ and computerized assessment were evaluated using intraclass correlation coefficients (ICC), Spearman rank correlation coefficients, or descriptive statistics. Results:Poor agreement among pathologists (ICC: 0.57) was found regarding the assessment of total steatosis, (ICC >0.7 indicates acceptable agreement). Pathologists’ estimation of micro- and macrosteatosis disclosed also poor correlation (ICC: 0.22, 0.55, respectively). Inconsistent assessment of histological features of steatohepatitis (lobular inflammation, portal inflammation, hepatocyte ballooning, and Mallory hyaline) was documented. Poor conformity was also shown between the computerized quantification and ratings of 3 pathologists (Spearman rank correlation coefficients: 0.22, 0.82, 0.28, and 0.38). Conclusion:Quantification of hepatic steatosis in histological sections is strongly observer-dependent, not reproducible, and does not correlate with the computerized estimation. Current standards of assessment, previously published data and the clinical relevance of hepatic steatosis for liver surgery and transplantation must be challenged.

Aggravation of viral hepatitis by platelet-derived serotonin.

More than 500 million people worldwide are persistently infected with hepatitis B virus or hepatitis C virus. Although both viruses are poorly cytopathic, persistence of either virus carries a risk of chronic liver inflammation, potentially resulting in liver steatosis, liver cirrhosis, end-stage liver failure or hepatocellular carcinoma. Virus-specific T cells are a major determinant of the outcome of hepatitis, as they contribute to the early control of chronic hepatitis viruses, but they also mediate immunopathology during persistent virus infection. We have analyzed the role of platelet-derived vasoactive serotonin during virus-induced CD8+ T cell–dependent immunopathological hepatitis in mice infected with the noncytopathic lymphocytic choriomeningitis virus. After virus infection, platelets were recruited to the liver, and their activation correlated with severely reduced sinusoidal microcirculation, delayed virus elimination and increased immunopathological liver cell damage. Lack of platelet-derived serotonin in serotonin-deficient mice normalized hepatic microcirculatory dysfunction, accelerated virus clearance in the liver and reduced CD8+ T cell–dependent liver cell damage. In keeping with these observations, serotonin treatment of infected mice delayed entry of activated CD8+ T cells into the liver, delayed virus control and aggravated immunopathological hepatitis. Thus, vasoactive serotonin supports virus persistence in the liver and aggravates virus-induced immunopathology.

Prevention of reperfusion injury and microcirculatory failure in macrosteatotic mouse liver by omega‐3 fatty acids

Macrovesicular hepatic steatosis has a lower tolerance to reperfusion injury than microvesicular steatosis with an abnormally high ratio of omega‐6 (n‐6): omega‐3 (n‐3) polyunsaturated fatty acids (PUFAs). We investigated the influence of PUFAs on microcirculation in steatotic livers and the potential to minimize reperfusion injury in the macrosteatotic liver by normalization of PUFAs. Ob/ob mice were used as a model of macrovesicular hepatic steatosis and C57/Bl6 mice fed a choline‐deficient diet for microvesicular steatosis. Steatotic and lean livers were subjected to 45 minutes of ischemia and 3 hours of reperfusion. Hepatic content of omega‐3 and omega‐6 PUFAs was determined. Microcirculation was investigated using intravital fluorescence microscopy. A second group of ob/ob mice was supplemented with dietary omega‐3 PUFAs and compared with the control diet–fed group. Microcirculation, AST, and Kupffer cell activity were assessed. Macrosteatotic livers had significant microcirculatory dysfunction correlating with high omega‐6: omega‐3 PUFA ratio. Dietary omega‐3 PUFA resulted in normalization of this ratio, reduction of intrahepatic lipids, and decrease in the extent of macrosteatosis. Defective microcirculation was dramatically ameliorated with significant reduction in Kupffer cell activity and protection against hepatocellular injury both before ischemia and after reperfusion. Conclusion: Macrosteatotic livers disclosed an abnormal omega‐6: omega‐3 PUFA ratio that correlates with a microcirculatory defect that enhanced reperfusion injury. Thus, protective strategies applied during or after ischemia are unlikely to be useful. Preoperative dietary omega‐3 PUFAs protect macrosteatotic livers against reperfusion injury and might represent a valuable method to expand the live liver donor pool. (HEPATOLOGY 2007;45:855–863.)

What is critical for liver surgery and partial liver transplantation: size or quality?

M ajor liver resections and partial orthotopic liver transplantation (OLT) have become established procedures in liver surgery; for many patients, these techniques offer the only curative option. Yet, many patients develop postoperative complications because the remnant livers or grafts are too small or of poor quality to sustain sufficient organ function. This somewhat new and poorly defined phenomenon has been termed ‘‘small-for-size syndrome’’ (SFSS) to describe this scenario. The concept is, in fact, not a new one, because as early as the 1970s, Thomas E. Starzl described the complicated postoperative course of a young woman subjected to an almost 90% hepatectomy and who was subsequently characterized by prolonged hyperbilirubinemia, encephalopathy, and coagulopathy. In an unconventional way for a review, we will start with three case reports to illustrate the scope and clinical relevance of SFSS after liver surgery and transplantation. Case 1: A 47-year-old healthy man, whose wife was listed for OLT due to a symptomatic nonresectable hemangioendothelioma of the liver, offered to be considered for living donor liver transplantation (LDLT). Following the standard work-up for this procedure, he underwent a right hemi-hepatectomy including the middle hepatic vein to serve as allograft for his wife. The remnant left hemi-liver was estimated by computed tomographic (CT) volumetry to weigh 450 g, i.e., around 32% of the whole liver. The ratio of the remnant liver weight to body weight (RLBW) was 0.65%. The donor had a difficult postoperative course developing mild encephalopathy and hyperbilirubinemia lasting 20 days peaking at 178 lmol/L (10.4 mg/ dL) by day five, and severe coagulopathy (prothrombin time <30%) that normalized by day 7. The donor eventually recovered fully, and was discharged in good general condition 22 days after surgery. Case 2: A 42-year-old male was listed for OLT because of Child B cirrhosis (Model for End-Stage Liver Disease [MELD] score: 21) and a small (3 cm) hepatocellular carcinoma (HCC) related to hepatitis B virus infection. He received the right hemi-liver containing the middle hepatic vein from his wife (graft weighing 620 g), who had an uneventful postoperative course. The ratio of graft size in grams to her husband’s body weight (80 kg) (graft-to-recipient weight ratio [GRWR]) was 0.7%. The postoperative period was complicated by encephalopathy, hyperbilirubinemia (up to 262 lmol/L, 15.3 mg/dL) for 2 weeks, and prolonged coagulopathy with a factor V level below 20% at day 4. As a result of the delayed graft function, the patient required intensive care unit treatment for 1 week before the liver graft function improved. He was able to be discharged in good general condition on postoperative day 21. Case 3: A 58-year-old male presented with multiple colorectal liver metastases in the right hemi-liver as well as in segment II, III, and 10 months after resection of the primary rectal tumor followed by 5 cycles of chemotherapy containing Folfox and Avastin. A Abbreviations: CALI, chemotherapy-associated liver injury; CT, computed tomography; DOI, 2,5-dimethoxy-4-iodoamphetamine; EHPBA, European Hepato-Pancreatico-Biliary Association; GRWR, graft-to-recipient weight ratio; HCC, hepatocellular carcinoma; IHPBA, International Hepato-PancreaticoBiliary Association; IL-6, interleukin-6; LDLT, living donor liver transplantation; MELD, Model for End-Stage Liver Disease; OLT, orthotopic liver transplantation; PTX, pentoxifylline; RLBW, remnant liver to body weight; SFSS, small-for-size syndrome; TNF, tumor necrosis factor. From the Swiss Hepato-Pancreatico-Biliary and Transplantation Center, Department of Surgery, University of Zurich, Zurich, Switzerland Funded in part by Grants from the Swiss National Foundation to P.A.C. (SNF 3200B0-109906), Krebsliga Zurich, Switzerland and Sassella Stiftung Zurich, Switzerland, also to P.A.C. Presented as a Thomas E. Starzl Transplant Surgery State-of-the-Art Lecture at the 60th Annual Meeting of the American Association for the Study of Liver Diseases; October 30-November 3, 2009; Boston, MA. This article is dedicated to Thomas E. Starzl for his lifelong contribution to liver surgery and transplantation. Address reprint requests to: Pierre-Alain Clavien, M.D., Ph.D., Department of Surgery, University Hospital of Zurich, Raemistrasse 100, 8091 Zurich, Switzerland. E-mail: clavien@access.uzh.ch; fax: þ41 44 255 44 49. CopyrightVC 2010 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hep.23713 Potential conflict of interest: Nothing to report.

Selective portal vein embolization and ligation trigger different regenerative responses in the rat liver

Two strategies are clinically available to induce selective hypertrophy of the liver: portal vein embolization (PVE) and portal vein ligation (PVL). The aim of this study was to compare the impact of PVE and PVL on liver regeneration. Rats were subjected to 70% PVL, 70% PVE, 70% partial hepatectomy (PH) (positive control), or sham operation (negative control). PVL and PVE of liver segments were validated by portography and histology, demonstrating obstruction of the involved portal branches. Liver weight and markers of regeneration were assessed at 24, 48, and 72 hours, and 7 days after surgery (n = 5). Sinusoidal perfusion was examined by intravital microscopy. The weight of the regenerating liver segments increased continuously in all groups, with the highest weight gain after PH, which also disclosed the strongest proliferative activity. In Ki‐67 and PCNA stainings, hepatocyte proliferation after PVL was more pronounced than after PVE (P = 0.01). Volumetric blood flow and functional sinusoidal density were lower after PVE than after PVL (P = 0.006, P = 0.02, respectively). The accumulation of Kupffer cells 24 hours after the intervention was highest after PH. Transcript levels of cytokines (interleukin‐1β, tumor necrosis factor‐α, interleukin‐6) peaked at 24 hours and were highest after PH. The embolized part of the liver after PVE showed prominent foreign body reaction in the portal triad with accumulation of macrophages. Conclusion: PVL is superior to PVE in inducing a regenerative response of the remnant liver. The impairment of liver regeneration after PVE may be a consequence of macrophage trapping in the occluded segment due to a foreign body reaction. Lower blood flow and lower accumulation of macrophages, particularly Kupffer cells, in the regenerating part of the liver likewise causes impaired liver regeneration after PVE. (HEPATOLOGY 2008.)

Activation of serotonin receptor-2B rescues small-for-size liver graft failure in mice.

The implantation of grafts below 30% of the normal liver volume is associated with a high risk of failure known as small‐for‐size (SFS) syndrome. Strategies to rescue small grafts may have a dramatic impact on organ shortage. Serotonin is a potent growth factor for the liver. The goal of this study was to determine whether enhanced serotonin signaling could prevent the deleterious effects of SFS syndrome. We performed 30% normal liver volume transplantations in wild‐type C57/BL6 and interleukin‐6 (IL‐6)−/− mice. Some animals received α‐methyl‐5‐HT (DOI), an agonist of serotonin receptor‐2 (5‐HT2B). Endpoints included long‐term survival, serum and hepatic markers of liver injury and regeneration, assessment of hepatic microcirculation by intravital fluorescence microscopy and scanning electron microscopy, and transcript levels of a variety of serotonin receptors, tumor necrosis factor α, and IL‐6. All recipients of small grafts (controls) died within 2‐4 days of transplantation, whereas half of those receiving DOI survived permanently. Control animals disclosed major liver injury, including diffuse microvesicular steatosis in hepatocytes, impairment of microcirculation, and a failure of regeneration, whereas these parameters were dramatically improved in animals subjected to DOI. Blockage of 5‐HT2B blunted the protective effects of DOI. Whereas IL‐6 levels were higher in DOI‐treated animals, IL‐6−/− mice were still protected by DOI, suggesting a protective pathway independent of IL‐6. Conclusion: Serotonin through its action on receptor‐2B protects SFS liver grafts from injury and prevents microcirculation and regeneration. The mechanism of hepato‐protection is independent of IL‐6. (Hepatology 2011;)

Low platelet counts after liver transplantation predict early posttransplant survival: The 60‐5 criterion

Platelets play a critical role in liver injury and regeneration. Thrombocytopenia is associated with increases in postoperative complications after partial hepatectomy, but it is unknown whether platelet counts could also predict outcomes after transplantation, a procedure that is often performed in thrombocytopenic patients. Therefore, the aim of this study was to evaluate whether platelet counts could be indicators of short‐ and long‐term outcomes after liver transplantation (LT). Two hundred fifty‐seven consecutive LT recipients (January 2003‐December 2011) from our prospective database were analyzed. Preoperative and daily postoperative platelet counts were recorded until postoperative day 7 (POD7). Univariate and multivariate analyses were performed to assess whether low perioperative platelet counts were a risk factor for postoperative complications and graft and patient survival. The median pretransplant platelet count was 88 × 109/L [interquartile range (IQR) = 58‐127 × 109/L]. The lowest platelet counts occurred on POD3: the median was 56 × 109/L (IQR = 41‐86 × 109/L). Patients with low platelet counts on POD5 had higher rates of severe (grade IIIb/IV) complications [39% versus 29%, odds ratio (OR) = 1.09 (95% CI = 1.1‐3.3), P = 0.02] and 90‐day mortality [16% versus 8%, OR = 2.25 (95% CI = 1.0‐5.0), P = 0.05]. In the multivariate analysis, POD5 platelet counts < 60 × 109/L were identified as an independent risk factor for grade IIIb/IV complications [OR = 1.96 (95% CI = 1.07‐3.56), P = 0.03)], graft survival [hazard ratio (HR) = 2.0 (95% CI = 1.1‐3.6), P = 0.03)], and patient survival [HR = 2.2 (95% CI = 1.1‐4.6), P = 0.03)]. The predictive value of platelet counts for graft and patient survival was lost in patients who survived 90 days. In conclusion, after LT, platelet counts < 60 × 109/L on POD5 (the 60‐5 criterion) are an independent factor associated with severe complications and early graft and patient survival. These findings may help us to develop protective strategies or specific interventions for high‐risk patients. Liver Transpl 20:147‐155, 2014.

Low Platelet Counts after Liver Transplantation Predict Early Post-Transplant Survival: The "60-5 Criterion"

Platelets play a critical role in liver injury and regeneration. Thrombocytopenia is associated with increases in postoperative complications after partial hepatectomy, but it is unknown whether platelet counts could also predict outcomes after transplantation, a procedure that is often performed in thrombocytopenic patients. Therefore, the aim of this study was to evaluate whether platelet counts could be indicators of short‐ and long‐term outcomes after liver transplantation (LT). Two hundred fifty‐seven consecutive LT recipients (January 2003‐December 2011) from our prospective database were analyzed. Preoperative and daily postoperative platelet counts were recorded until postoperative day 7 (POD7). Univariate and multivariate analyses were performed to assess whether low perioperative platelet counts were a risk factor for postoperative complications and graft and patient survival. The median pretransplant platelet count was 88 × 109/L [interquartile range (IQR) = 58‐127 × 109/L]. The lowest platelet counts occurred on POD3: the median was 56 × 109/L (IQR = 41‐86 × 109/L). Patients with low platelet counts on POD5 had higher rates of severe (grade IIIb/IV) complications [39% versus 29%, odds ratio (OR) = 1.09 (95% CI = 1.1‐3.3), P = 0.02] and 90‐day mortality [16% versus 8%, OR = 2.25 (95% CI = 1.0‐5.0), P = 0.05]. In the multivariate analysis, POD5 platelet counts < 60 × 109/L were identified as an independent risk factor for grade IIIb/IV complications [OR = 1.96 (95% CI = 1.07‐3.56), P = 0.03)], graft survival [hazard ratio (HR) = 2.0 (95% CI = 1.1‐3.6), P = 0.03)], and patient survival [HR = 2.2 (95% CI = 1.1‐4.6), P = 0.03)]. The predictive value of platelet counts for graft and patient survival was lost in patients who survived 90 days. In conclusion, after LT, platelet counts < 60 × 109/L on POD5 (the 60‐5 criterion) are an independent factor associated with severe complications and early graft and patient survival. These findings may help us to develop protective strategies or specific interventions for high‐risk patients. Liver Transpl 20:147‐155, 2014.

The effect of perfluorocarbon-based artificial oxygen carriers on tissue-engineered trachea.

The biological effect of the perfluorocarbon-based artificial oxygen carrier (Oxygent) was investigated in tissue-engineered trachea (TET) construction. Media supplemented with and without 10% Oxygent were compared in all assessments. Partial tissue oxygen tension (PtO(2)) was measured with polarographic microprobes; epithelial metabolism was monitored by microdialysis inside the TET epithelium perfused with the medium underneath. Chondrocyte-DegraPol constructs were cultured for 1 month with the medium before glycosaminoglycan assessment and histology. Tissue reaction of TET epithelial scaffolds immersed with the medium was evaluated on the chick embryo chorioallantoic membrane. Oxygent perfusion medium increased the TET epithelial PtO(2) (51.2 +/- 0.3 mm Hg vs. 33.4 +/- 0.3 mm Hg at 200 microm thickness; 12.5 +/- 0.1 mm Hg vs. 3.1 +/- 0.1 mm Hg at 400 microm thickness, p < 0.01) and decreased the lactate concentration (0.63 +/- 0.08 vs. 0.80 +/- 0.06 mmol/L, p < 0.05), lactate/pyruvate (1.87 +/- 0.26 vs. 3.36 +/- 10.13, p < 0.05), and lactate/glucose ratios (0.10 +/- 0.00 vs. 0.29 +/- 0.14, p < 0.05). Chondrocyte-DegraPol in Oxygent group presented lower glycosaminoglycan value (0.03 +/- 0.00 vs. 0.13 +/- 0.00, p < 0.05); histology slides showed poor acid mucopolysaccharides formation. Orthogonal polarization spectral imaging showed no difference in functional capillary density between the scaffolds cultured on chorioallantoic membranes. The foreign body reaction was similar in both groups. We conclude that Oxygent increases TET epithelial PtO(2), improves epithelial metabolism, does not impair angiogenesis, and tends to slow cartilage tissue formation.

Chemical composition of hepatic lipids mediates reperfusion injury of the macrosteatotic mouse liver through thromboxane A2

BACKGROUND & AIMS Chemical composition of hepatic lipids is an evolving player in steatotic liver ischemia/reperfusion (I/R) injury. Thromboxane A(2) (TXA(2)) is a vasoactive pro-inflammatory lipid mediator derived from arachidonic acid (AA), an omega-6 fatty acid (Ω-6 FA). Reduced tolerance of the macrosteatotic liver to I/R may be related to increased TXA(2) synthesis due to the predominance of Ω-6 FAs. METHODS TXA(2) levels elicited by I/R in ob/ob and wild type mice were assessed by ELISA. Ob/ob mice were fed Ω-3 FAs enriched diet to reduce hepatic synthesis of AA and TXA(2) or treated with selective TXA(2) receptor blocker before I/R. RESULTS I/R triggered significantly higher hepatic TXA(2) production in ob/ob than wild type animals. Compared with ob/ob mice on regular diet, Ω-3 FAs supplementation markedly reduced hepatic AA levels before ischemia and consistently blunted hepatic TXA(2) synthesis after reperfusion. Sinusoidal perfusion and hepatocellular damage were significantly ameliorated despite downregulation of heme oxygenase-1. Hepatic transcript and protein levels of IL-1β and neutrophil recruitment were significantly diminished after reperfusion. Moreover, TXA(2) receptor blockage conferred similar protection without modification of the histological pattern of steatosis. A stronger protection was achieved in the steatotic compared with lean animals. CONCLUSIONS Enhanced I/R injury in the macrosteatotic liver is explained, at least partially, by TXA(2) mediated microcirculatory failure rather than size-related mechanical compression of the sinusoids by lipid droplets. TXA(2) blockage may be a simple strategy to include steatotic organs and overcome the shortage of donor organs for liver transplantation.