Linda D. Ferrell

Design and validation of a histological scoring system for nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is characterized by hepatic steatosis in the absence of a history of significant alcohol use or other known liver disease. Nonalcoholic steatohepatitis (NASH) is the progressive form of NAFLD. The Pathology Committee of the NASH Clinical Research Network designed and validated a histological feature scoring system that addresses the full spectrum of lesions of NAFLD and proposed a NAFLD activity score (NAS) for use in clinical trials. The scoring system comprised 14 histological features, 4 of which were evaluated semi‐quantitatively: steatosis (0‐3), lobular inflammation (0‐2), hepatocellular ballooning (0‐2), and fibrosis (0‐4). Another nine features were recorded as present or absent. An anonymized study set of 50 cases (32 from adult hepatology services, 18 from pediatric hepatology services) was assembled, coded, and circulated. For the validation study, agreement on scoring and a diagnostic categorization (“NASH,” “borderline,” or “not NASH”) were evaluated by using weighted kappa statistics. Inter‐rater agreement on adult cases was: 0.84 for fibrosis, 0.79 for steatosis, 0.56 for injury, and 0.45 for lobular inflammation. Agreement on diagnostic category was 0.61. Using multiple logistic regression, five features were independently associated with the diagnosis of NASH in adult biopsies: steatosis (P = .009), hepatocellular ballooning (P = .0001), lobular inflammation (P = .0001), fibrosis (P = .0001), and the absence of lipogranulomas (P = .001). The proposed NAS is the unweighted sum of steatosis, lobular inflammation, and hepatocellular ballooning scores. In conclusion, we present a strong scoring system and NAS for NAFLD and NASH with reasonable inter‐rater reproducibility that should be useful for studies of both adults and children with any degree of NAFLD. NAS of ≥5 correlated with a diagnosis of NASH, and biopsies with scores of less than 3 were diagnosed as “not NASH.” (HEPATOLOGY 2005;41:1313–1321.)

HCV-related fibrosis progression following liver transplantation: increase in recent years

BACKGROUND/AIMS The natural history and predictors of HCV-related disease severity post-transplantation are uncertain. The aims of this study were to define the natural history of post-transplantation HCV infection by assessing the rate of fibrosis progression, to determine if the post-transplantation natural history differs from that observed pre-transplantation, and to identify predictors of post-transplantation disease progression. METHODS Post-transplantation biopsies (mean: 3+/-1.6/patient) from 284 patients were scored according to histologic stage, using the method of Desmet et al. Change in fibrosis score (fibrosis progression/year) post-transplantation was used as the primary outcome. Predictors analyzed included viral factors (genotype and viral load at transplantation), patient demographics, year of transplantation, country of transplantation, pre-transplantation fibrosis progression, immunosuppression and laboratory data. RESULTS There was a linear association between change in fibrosis score and time from transplantation, with a median rate of fibrosis progression per year of 0.3 (0.004-2.19/year). Using parametric time-to-event analysis, the expected median duration to cirrhosis was 10 years. The rate of post-transplantation fibrosis progression was significantly higher than pre-transplantation (0.2/year (0.09-0.8) p<0.0001), and higher in Spanish than US centers (0.48 (0.01-2.19) vs 0.28 (0.004-2.08); p=0.09) despite similar progression rates prior to transplantation. Variables independently associated with post-transplantation progression included year of transplantation (p=0.0001), race (p=0.02), number of methyl-prednisolone boluses (p=0.03), and HCV RNA levels at transplantation (p=0.01). CONCLUSIONS HCV-related disease progression is accelerated in immunocompromised compared to immunocompetent patients, with a progressive increase in patients who have recently undergone liver transplantation. Changes in patient management post-transplantation over time and between transplant centers may account for the increase in fibrosis progression observed in recent years.

Functional analysis of grafts from living donors. Implications for the treatment of older recipients.

OBJECTIVE Living-related liver transplantation (LRLT) has established efficacy in children. In a larger recipient, LRLT requires the use of a small graft because of limits on the donor hepatectomy. SUMMARY BACKGROUND DATA The minimum graft weight required for successful transplantation has not been well established, although a characteristic pattern of graft dysfunction has been observed in our patients who receive small grafts. The authors present a clinicopathologic study of small liver grafts obtained from living donors. METHODS Clinical and histologic data were reviewed for 25 patients receiving LRLT. In five older recipients (small group), the graft represented 50% or less of expected liver weight, whereas in 20 others (large group), the graft represented at least 60% of expected liver weight. A retrospective analysis of graft function was conducted by analyzing clinical parameters and histology. RESULTS In the small group, 2 of 5 grafts (40%) were lost due to poor function, leading to one patient death (20% mortality), whereas in the large group, 2 of 20 grafts (10%) were lost due to arterial thrombosis without patient mortality. Early ischemic damage related to transplant was comparable with aspartate aminotransferase 203 +/- 23 (small group) and 290 +/- 120 (large group) at 24 hours (p = not significant). Early function was significantly decreased in the small group, with prothrombin time 18.2 +/- 2.2 seconds versus 14.8 +/- 1.6 seconds (large group) on day 3 (p = 0.034). All small group patients developed cholestasis with significantly increased total bilirubin levels at day 7 (16 +/- 5.2 mg% vs. 3.7 +/- 2.7 mg%; p = 0.021) and day 14 (12.0 +/- 7.4 vs. 1.8 +/- 0.7; p = 0.021) compared with the large group. Protocol biopsies in the small group revealed a diffuse ischemic pattern with cellular ballooning on day 7, which progressed to cholestasis in subsequent biopsies. Large group biopsies showed minimal ischemic changes. Three small group patients recovered with normal liver function by 12 weeks. CONCLUSIONS Clinical recovery after a small-for-size transplant is characterized by significant functional impairment associated with paradoxical histologic changes typical of ischemia. These changes apparently are due to graft injury, which can only be the result of small graft size. These findings have significant implications for the extension of LRLT to adults.

Carotid plaque histology using real-time ultrasonography: Clinical and therapeutic implications☆☆☆

To evaluate the ability of ultrasonographic imaging to detect plaque hemorrhage in carotid atheroma, a study was undertaken that compared pathologic findings to preoperative ultrasonographic findings. Ultrasonography identified two plaque categories based on the heterogeneous and homogeneous echo patterns of the lesions studied. Heterogeneous lesions accounted for 91 percent of intraplaque hemorrhages (30 of 33) and 100 percent of ulcerated lesions (15 of 15). In 41 of 50 specimens (82 percent), ultrasonography correctly identified the presence or absence of plaque hemorrhage. False-negative studies (3 of 50) were due to the minute foci of remote hemorrhages. False-positive studies (6 of 50) resulted from plaques that contained large amounts of lipid or cholesterol. Preoperative ultrasound carotid imaging can be used to detect the histologic characteristics of plaque. Since recent clinicopathologic studies have implicated intraplaque hemorrhage and ulceration in symptomatic carotid disease, this information may be of value in choosing therapy, especially for the asymptomatic patient.

Hepatic angiomyolipoma - A clinicopathologic study of 30 cases and delineation of unusual morphologic variants

Hepatic angiomyolipoma (AML) is frequently misdiagnosed. HMB-45 is a promising immunomarker for this tumor that leads to recognition of some AMLs with unusual morphology. The purpose of this collaborative study is to better define the morphologic variations of AML. Thirty AMLs were examined, including four biopsy specimens and two fine-needle aspirates. The diagnosis was confirmed by the presence of HMB-45-positive myoid cells. Almost half the cases were originally misdiagnosed as carcinomas or sarcomas. There was marked female predominance (25:5), and the mean age was 48.7 years (range 29-68). Three patients (10%) had evidence of tuberous sclerosis and all had renal AML. According to the line of differentiation and predominance of tissue components, the tumors was subcategorized into mixed, lipomatous (> or = 70% fat), myomatous (< or = 10% fat), and angiomatous type. The mixed type was the most common (11 resected cases), comprising sheets of epithelioid muscle cells admixed with islands of adipocytes, abnormal vessels, and frequently, hematopoietic cells. Six tumors (including three from biopsy specimens) were heavily fatty and showed predominantly adipocytes with epithelioid and short spindle myoid cells webbed between fat cells. Of 10 myomatous AMLs, five tumors showed a pure sinusoidal trabecular pattern and comprised mainly epithelioid cells. Typically, mature adipocytes were absent or scanty, but fat was seen as fine droplets within cytoplasm or as occasional large globules in sinusoids. Pelioid and inflammatory pseudotumor-like patterns were identified focally. Regarding cellular features of the myoid cells, most of the epithelioid cells were either eosinophilic or clear with spiderweb cell morphology. Three AMLs showed an almost purely oncocytic appearance with scanty fat. Large pleomorphic epithelioid cells existed as small foci. Spindle cells arranged in long fascicles were uncommon. D-PAS-positive globules were common around pelioid areas. Brown pigments with staining characteristics of hemosiderin and/or melanin were noted. In conclusion, we propose HMB-45-positive myoid cells as the defining criterion of hepatic AML, which is a tumor capable of dual myomatous and lipomatous differentiation and melanogenesis. Because of its protean morphologic appearance, recognition of the various variant patterns and cell types is important for a correct diagnosis, assisted by immunohistochemical confirmation with HMB-45. Trabecular and oncocytic cell tumors appear to stand out as distinctive subtypes.

Caerulein-induced acute necrotizing pancreatitis in mice: protective effects of proglumide, benzotript, and secretin.

The onset, course, and regression of the biochemical and structural alterations associated with pancreatitis induced by various doses of caerulein were studied in the mouse. In addition, the protective effect of secretin was compared with that of the cholecystokinin-receptor antagonists proglumide and benzotript. Subcutaneous or intraperitoneal injections of caerulein induced increases in serum amylase concentration and pancreatic weight and histologic evidence of acute pancreatitis, all effects being dose-related. Cytoplasmic vacuoles were the earliest histologic alterations. As the pancreatitis progressed these vacuoles increased to an enormous size. Interstitial inflammation and acinar cell necrosis were prominent after 6 h, reached a maximum after 12 h, and mostly disappeared after 4 days. During the course of pancreatitis approximately 40% of the acinar cells showed signs of severe degeneration or necrosis at the most effective doses of caerulein. Electron microscopy showed both intact and degenerating granules inside the vacuoles. Signs of basolateral exocytosis of zymogen granules were not observed. During the regression of pancreatitis, focal atrophy was a remarkable histologic finding. Repetitive initiation of pancreatitis (six courses of caerulein injections over 5 wk) produced marked focal atrophy and early fibrosis. High doses of proglumide or benzotript markedly ameliorated both the biochemical and structural alterations induced by caerulein. Secretin, even at very high doses, had only minor protective effects. This study presents a model of acute necrotizing pancreatitis in which the severity of the induced pancreatitis ranges dose-dependently from mild interstitial inflammation to severe necrosis. The ultrastructural alterations described herein support the hypothesis that the trigger mechanism of acute pancreatitis appears to be a primary intracellular event rather than an interstitial event that secondarily damages the acinar cells.

Occult hepatitis B virus as source of infection in liver transplant recipients

Hepatitis B virus (HBV) infection almost always recurs after liver transplantation in patients who were surface antigen (HBsAg) positive before surgery but apparent de novo acquisition of infection in a transplant setting has not previously been reported. We have used sensitive techniques to elucidate the origin of such infections in patients in a California transplantation programme. We tested post-transplant serum from 207 patients who had been HBsAg negative and found 20 to be HBsAg positive. The origin of infection was identified in 7 patients, being occult pre-transplant infection in 5 and occult infection in the donor in 2. No pre-transplant patient nor donor with demonstrable HBV DNA had serological markers of hepatitis B. Post-transplant HBV DNA was present in serum from 19 patients. Analysis of the variable pre-S region of HBV demonstrated 100% sequence homology between recipient liver and post-transplant serum (2 patients) and between donor serum and recipient post-transplant serum (2). There was only 84% homology between the 2 different patients infected with subtype adw. 19 patients are alive, 9 without histological evidence of hepatitis (mean follow-up 33 months), and survival was significantly greater than that of a group with recurrent HBV infection. Apparent acquisition of HBV infection with liver transplantation is not rare, and may be due to occult pre-transplant infection or occult infection in the donor. The post-transplant outcome of this infection tends to be benign but our findings do underscore the clinical relevance of HBV infection in the absence of serological markers.

Correlation of paired liver biopsies in morbidly obese patients with suspected nonalcoholic fatty liver disease

In the absence of surrogate markers, the evaluation of suspected nonalcoholic fatty liver disease (NAFLD) is highly dependent on histological examination. The extent of sampling variability affecting the reliability of a single liver biopsy in patients with suspected NAFLD is poorly characterized. This prospective study aimed to correlate precise histological findings in paired biopsies—right and left lobe—in the diagnosis of NAFLD in morbidly obese subjects undergoing bariatric surgery employing both Brunt and Matteoni classifications and the NAFLD Activity Score (NAS). We also aimed to determine whether the composite histopathological findings of the two biopsies would improve diagnostic accuracy. Consecutive subjects had an intraoperative biopsy from both right and left lobes, evaluated and scored in a blinded manner. Intraobserver agreement was also assessed. Kappa coefficients of agreement were calculated. Forty‐one subjects had acceptable biopsies. Agreement for steatosis was excellent and moderate for fibrosis. Concordance was only fair for most features of necroinflammation. Intraobserver agreement was only moderate for lobular inflammation. Excellent agreement was seen for the diagnosis of NASH using Brunt criteria and good agreement when using Matteoni and NAS scoring systems. Composite biopsy data particularly improved identification of hepatocyte ballooning. The diagnostic accuracy also improved substantially when composite features were compared with single‐sided biopsy features, especially for the Matteoni and NAS scoring systems. In conclusion, significant sampling variability occurs in NAFLD, particularly for features of necroinflammation. This should be factored into the design of clinical trials and studies of the natural history of the disease. (HEPATOLOGY 2006;44:874–880.)

Pancreatic exocrine secretion in acute experimental pancreatitis

Little is known about exocrine pancreatic secretory function in patients with acute pancreatitis, in particular during the early phase of the disease. Therefore, this study evaluates basal and stimulated pancreatic secretion in vivo and in vitro in four different models of acute pancreatitis which reflect its clinical spectrum of severity: (a) edematous pancreatitis induced in the rat by seven IP injections of 50 micrograms/kg cerulein at hourly intervals; (b) edematous pancreatitis with cellular necrosis induced in the mouse by seven IP injections of 50 micrograms/kg cerulein at hourly intervals; (c) hemorrhagic pancreatitis induced in the mouse by feeding an ethionine-supplemented, choline-deficient diet for 66 hours; and (d) hemorrhagic pancreatitis induced in the rat by retrograde infusion of 0.6 mL 5% sodium taurocholate into the pancreatic duct. Secretory studies were performed in vivo and in vitro at various times after onset of pancreatitis. The results show that the exocrine pancreas gradually became resistant to cholecystokinin stimulation after the onset of acute pancreatitis in all four animal models. Cholecystokinin-stimulated secretion was almost abolished in vivo and in vitro at the time of maximal histological damage. In vivo basal secretion was also reduced. In vitro there was an increase in basal release of amylase from isolated acini that was not caused by an increase in luminal secretion but by enzyme release from damaged cells. The time course of improvement of secretory function after acute experimental pancreatitis depended on the severity of the pancreatitis. Recovery of secretory capacity took longer after severe necrotizing pancreatitis than after edematous pancreatitis. However, the ultimate resolution of secretory function was remarkable, in particular after severe hemorrhagic pancreatitis. In all four models, secretory capacity became indistinguishable from normal before the morphological alterations had completely resolved. The present experimental data suggest that pancreatic secretion, and particularly pancreatic secretory response to cholecystokinin, may also be reduced in patients early after the onset of acute pancreatitis.

Biliary intraepithelial neoplasia: an international interobserver agreement study and proposal for diagnostic criteria

Cholangiocarcinoma of the intrahepatic and extrahepatic bile ducts develops through a multistep histopathologic sequence. Premalignant or non-invasive neoplastic lesions of bile ducts have been historically called biliary dysplasia or atypical biliary epithelium. To this date, no standard terminology or classification system has been offered for these lesions. In 2005, a conceptual framework and diagnostic criteria for biliary intraepithelial neoplasia (BilIN) were proposed using the livers of patients with hepatolithiasis. We report herein an international interobserver agreement study on the diagnosis of biliary non-invasive neoplastic lesions with the goal to obtain a consensus on the terminology and grading. Seventeen pathologists from the United States, Europe and Asia participated in this study. They shared a digital file containing histological pictures of 30 foci of non-invasive neoplastic lesions selected from the biliary system of patients suffering from primary sclerosing cholangitis, choledochal cyst or hepatolithiasis. In the criteria, we proposed in 2005, BilIN was classified into three categories based on the degree of atypia: BilIN-1, BilIN-2 and BilIN-3. In this study, consensus was reached for the terminology of BilIN and the three-grade classification system. Interobserver agreement on the diagnosis was moderate (κ-value=0.45). On the basis of the suggestions and opinions obtained from the 17 participants, the original criteria for BilIN were revised. We now propose a new consensus classification of BilIN that may assist in allowing a more uniform terminology for the diagnosis of biliary non-invasive neoplastic lesions. This classification should help to advance clinical and research applications.