Diagnostic histochemistry in hepatic pathology.

Histochemistry has an important, continuing role in the current assessment of hepatic biopsies and resection specimens. The evaluation of connective tissue elements in the liver can be accomplished with such methods as the Masson trichrome, Snook reticulin, Vierhoff van Gieson, orcein, and Victoria blue stains. The results contribute to the diagnosis of acute and chronic hepatitis, submassive necrosis, venous outflow obstruction, steatohepatitis, and cirrhosis. Fat stains done on frozen sections of liver tissue are routinely performed in the evaluation of donor liver allograft biopsies. Iron stains such as Perls' method and the Prussian blue technique contribute to the recognition of hemochromatosis and hemosiderosis. The rhodanine, orcein, and Timm stains for copper are used in the characterization of chronic cholestatic liver disease and Wilson's disease. Labeling of carbohydrate-based moieties in various disorders is accomplished with the digested and undigested periodic acid-Schiff method, and Congo red or crystal violet stains can be employed to detect amyloid deposition. Lastly, evaluations of the thickness of the cell plates and continuity of the reticulin framework, as seen with the Snook reticulin stain, can contribute to the diagnostic separation of benign from malignant hepatocellular neoplasms.

Chronically inflamed livers up-regulate expression of inhibitory B7 family members.

UNLABELLED: Hepatitis B virus, hepatitis C virus, autoimmune hepatitis, and nonalcoholic fatty liver disease can induce chronic liver disease. The Programmed Death-1 (PD-1) inhibitory pathway assists in T cell response regulation during acute and chronic inflammation and participates in the progression of inflammatory liver disease. To examine whether PD-1 and its ligands, B7-H1 and B7-DC, are modulated during chronic necroinflammatory liver disease, we investigated expression profiles in normal patients and patients with the aforementioned conditions. Relative to liver biopsies from normal individuals, those from patients with chronic necroinflammatory liver diseases (hepatitis B virus, hepatitis C virus, and autoimmune hepatitis) contain increased numbers of PD-1-expressing lymphocytes. Kupffer cells, liver sinusoidal endothelial cells, and leukocytes express PD-1 ligands. We also detect PD-1 ligands on hepatocytes within biopsies and on isolated cells. All forms of chronic necroinflammatory liver disease examined correlate with increased B7-H1 and B7-DC expression on Kupffer cells, liver sinusoidal epithelial cells, and leukocytes. The degree of necroinflammation correlates with expression levels of PD-1 family members. CONCLUSION: These results demonstrate that expression of PD-1/PD-1 ligands links more directly with the degree of inflammation than with the underlying etiology of liver damage. The PD-1 pathway may assist the liver in protecting itself from immune-mediated destruction.

Thiazolidinediones for the treatment in NASH: sustained benefit after drug discontinuation?

BACKGROUND: Although of unproven benefit for nonalcoholic steatohepatitis (NASH), thiazolidinediones (TZDs) have emerged as a promising therapy. Little evidence exists, however, regarding sustained effects of TZDs in NASH after drug discontinuation. A recent clinical study suggests that relapse of NASH pathophysiology is inevitable and that lifelong therapy may be needed to maintain histologic response. GOAL: We reviewed extended follow-up data of NASH patients previously treated with troglitazone to evaluate the influence of weight and physical activity on clinical and histologic parameters related to NASH recurrence. STUDY: After medical record review, 9 of 10 patients had complete data for follow-up and 5 had an extended follow-up biopsy 3 years or more after discontinuing troglitazone therapy. RESULTS: In contrast to recent work showing relapse of NASH to be nearly universal after discontinuation of TZD therapy, 2 of our patients had 1-stage improvement in fibrosis, normal aminotransferases, and absence of diabetes after median follow-up of 37 months after discontinuation of troglitazone. Both patients had sustained exercise programs and interval body mass index reduction. In contrast, active steatohepatitis, progression of fibrosis, and requirement of antidiabetic medications were seen in patients unable to achieve lifestyle modifications. CONCLUSIONS: We conclude that sustained histologic response after short-term TZD therapy for NASH is not invariably lost after medication discontinuation but rather is intimately related to sustained changes in lifestyle, particularly physical activity. Activity and diet in the setting of thiazolidinedione or other drug therapy of NASH is an essential consideration that warrants careful incorporation into future drug trials.

Report and Recommendations of the Association of Pathology Chairs' Autopsy Working Group.

Autopsy has been a foundation of pathology training for many years, but hospital autopsy rates are notoriously low. At the 2014 meeting of the Association of Pathology Chairs, some pathologists suggested removing autopsy from the training curriculum of pathology residents to provide additional months for training in newer disciplines, such as molecular genetics and informatics. At the same time, the American Board of Pathology received complaints that newly hired pathologists recently certified in anatomic pathology are unable to perform an autopsy when called upon to do so. In response to a call to abolish autopsy from pathology training on the one hand and for more rigorous autopsy training on the other, the Association of Pathology Chairs formed the Autopsy Working Group to examine the role of autopsy in pathology residency training. After 2 years of research and deliberation, the Autopsy Working Group recommends the following:Autopsy should remain a component of anatomic pathology training.A training program must have an autopsy service director with defined responsibilities, including accountability to the program director to record every autopsy performed by every resident.Specific entrustable activities should be defined that a resident must master in order to be deemed competent in autopsy practice, as well as criteria for gaining the trust to perform the tasks without direct supervision.Technical standardization of autopsy performance and reporting must be improved.The current minimum number of 50 autopsies should not be reduced until the changes recommended above have been implemented.

"Undifferentiated" small round cell tumors of the sinonasal tract: differential diagnosis update.

Sinonasal tract neoplasms composed of light microscopically seemingly "undifferentiated" small round cells often generate considerable diagnostic difficulty. Although the careful review of H&E-stained sections remains of critical and central importance in this evaluation, the recent improvements in the immunohistochemical diagnostic armamentarium and molecular diagnostic techniques applicable to paraffin-embedded tissue samples may add diagnostically valuable information. Accordingly, this review will discuss the differential diagnosis of undifferentiated small blue cell tumors of the sinonasal tract based on the light microscopic and clinical features and, as needed, the results of these ancillary studies. Tumors discussed include olfactory neuroblastoma, sinonasal undifferentiated carcinoma, small cell undifferentiated (neuroendocrine) carcinoma, undifferentiated (lymphoepithelioma-like) carcinoma, malignant melanoma, pituitary adenoma, Ewing sarcoma/peripheral neuroectodermal tumor, rhabdomyosarcoma, mesenchymal chondrosarcoma, small cell osteosarcoma, synovial sarcoma, extranodal natural killer/T-cell lymphoma, nasal type, and extramedullary plasmacytoma.

Biomechanical properties of tissue-engineered cartilage from human and rabbit chondrocytes.

OBJECTIVE: To describe tissue-engineered cartilage from rabbit and human chondrocytes. STUDY DESIGN AND SETTING: Chondrocytes from rabbit and human ears were seeded onto a template and implanted for 8 or 16 weeks of in vivo incubation. RESULTS: For the 8-week and 16-week groups, the UTS for cartilage was 3.8 MPa and 3.7 MPa, stiffness was 62.4 MPa and 51.8 MPa, and resilience was 181.8 J/m(3) and 109.1 J/m(3), respectively. Experimental cartilage was significantly different from controls. From 5 human specimens, the UTS was 5.4 MPa, stiffness was 6.6 MPa, and resilience was 2.0 J/m(3). The control had UTS of 8.8 MPa, stiffness of 12.2 MPa, and resilience of 2.9 J/m(3). Histology showed mature cartilage but with a fibrovascular infiltrate and increased cellularity. CONCLUSIONS: Mechanical properties of tissue-engineered cartilage can be quantified and are less than that of controls.

Effects of transforming growth factor Beta and insulinlike growth factor 1 on the biomechanical and histologic properties of tissue-engineered cartilage.

OBJECTIVE: To investigate the histologic and biomechanical properties of rabbit tissue-engineered cartilage exposed to insulinlike growth factor 1 and transforming growth factor beta. DESIGN: Controlled study. SUBJECTS: New Zealand white rabbits aged 3 to 4 weeks. INTERVENTION: A mean of 3.42 million rabbit chondrocytes were placed onto 2 x 1-cm polyglycolic/poly-L-lactic acid mesh templates. One group (n = 21) was placed in complete medium for 4 days. The experimental group (n = 19) was placed into complete medium with insulinlike growth factor 1 (50 ng/mL) and transforming growth factor beta (1 ng/mL). After 96 hours the templates were removed and implanted into the dorsum of the donor rabbit. The templates were harvested after 8 weeks and subjected to gross, histologic, and biomechanical testing. RESULTS: All samples showed histologic characteristics consistent with normal cartilage. No statistically significant differences were found with biomechanical testing between the control and experimental groups. CONCLUSION: In spite of more promising results from earlier studies, these results do not support improved histologic features or mechanical performance with the addition of insulinlike growth factor 1 and transforming growth factor beta to the chondrocyte/template complex.

Selective pathology fellowships: diverse, innovative, and valuable subspecialty training.

CONTEXT: Although selective pathology fellowships have a long-standing history of developing trainees with advanced expertise in specific areas of pathology other than those of the American Board of Pathology-certified subspecialties, the widespread interest in this training continues to grow. OBJECTIVE: To describe the historical background and current status of selective pathology fellowships, and to provide examples of 3 programs. In addition, Accreditation Council for Graduate Medical Education (ACGME)-accredited programs and nonaccredited programs in Selective Pathology are compared. DATA SOURCES: ACGME data banks and publicly available online materials were used. Program directors of the fellowships examples in this paper provided program-specific information. Additionally, an online survey of the program directors and program coordinators of ACGME-accredited programs and nonaccredited programs in selective pathology was performed. CONCLUSIONS: There are currently 76 ACGME-accredited selective pathology programs. The programs are distributed between 3 major categories: surgical pathology, focused anatomic pathology, and focused clinical pathology. Although the vast majority of programs are concerned that their funding source may be cut in the next 3 years, most programs will not change the number of fellowship positions in their programs. Program requirements devoted specifically and solely to selective pathology have been developed and are in effect. The value of this training is recognized not only by pathologists, but by clinicians as well, in both academia and private practice. Importantly, the diversity and innovation inherent in selective pathology allow these programs to adeptly address new subspecialty areas and technologic advances in the current and evolving practice of pathology.

The pathology milestones and the next accreditation system.

CONTEXT: In the late 1990s, the Accreditation Council for Graduate Medical Education developed the Outcomes Project and the 6 general competencies with the intent to improve the outcome of graduate medical education in the United States. The competencies were used as the basis for developing learning goals and objectives and tools to evaluate residents' performance. By the mid-2000s the stakeholders in resident education and the general public felt that the Outcomes Project had fallen short of expectations. OBJECTIVE: To develop a new evaluation method to track trainee progress throughout residency using benchmarks called milestones. A change in leadership at the Accreditation Council for Graduate Medical Education brought a new vision for the accreditation of training programs and a radically different approach to the evaluation of residents. DATA SOURCES: The Pathology Milestones Working Group reviewed examples of developing milestones in other specialties, the literature, and the Accreditation Council for Graduate Medical Education program requirements for pathology to develop pathology milestones. The pathology milestones are a set of objective descriptors for measuring progress in the development of competency in patient care, procedural skill sets, medical knowledge, practice-based learning and improvement, interpersonal and communication skills, professionalism, and systems-based practice. CONCLUSIONS: The milestones provide a national standard for evaluation that will be used for the assessment of all residents in Accreditation Council for Graduate Medical Education-accredited pathology training programs.

Hepatocyte cytokeratin 7 expression in chronic allograft rejection.

We examined hepatocyte cytokeratin 7 (CK7) expression in chronic allograft rejection (CR), a ductopenic condition in which this has not been systematically evaluated, in 20 patients with the clinicopathologic diagnosis of CR and age-, sex-, and native-disease-matched control subjects. We also studied baseline biopsy specimens from both groups. Three pathologists independently reviewed H&E- and CK7-stained sections, counting interlobular bile ducts (BDs) and portal tracts (PTs), noting the morphologic pattern of injury and scoring hepatocyte CK7 expression (0, none; 1+, rare; 2+, multifocal, predominantly periportal; 3+, extension into the lobule; 4+, diffuse). Mean BD/PT ratios and CK7 scores were calculated. The mean BD/PT ratio (0.58) and CK7 score (1.01) for the "CR, diagnostic" group were significantly different from all other group means (P < .05); no other comparisons were significant (P > .05). A CK7 score of 1 or more was observed in 9 (56%) of 16 CR specimens and in 3 (7%) of 41 remaining specimens. Hepatocyte CK7 expression is frequently noted in CR, and it would appear to reflect ductopenia. CK7 staining may be a useful diagnostic adjunct in evaluation of transplant liver biopsy specimens.

Expression of perilipin and adipophilin in nonalcoholic fatty liver disease; relevance to oxidative injury and hepatocyte ballooning.

AIMS: Perilipin and adipophilin, PAT family proteins, play important roles in lipid metabolism. Although nonalcoholic fatty liver disease (NAFLD) is initiated by hepatocyte lipidation, little is known about the relationship between these proteins and hepatocellular injury. We investigated the expressions of perilipin and adipophilin and their relation to inflammation, fibrosis, hepatocellular ballooning, and oxidized phosphatidylcholine (oxPC) localization in human NAFLD. METHODS AND RESULTS: Liver biopsies of nonalcoholic steatohepatitis (NASH, n=39) or simple steatosis (n=9) were studied by immunohistochemical techniques using anti-perilipin, anti-adipophilin and anti-oxPC antibodies. The severity of liver damage was histologically assessed by the Brunt system and NAFLD activity score (NAS). Enlarged hepatocytes usually containing Mallory-Denk bodies were defined as ballooned. Perilipin and adipophilin were detected on the rim of lipid droplets in both NASH and simple steatosis. Perilipin was more evident in larger lipid droplets while adipophilin expression was frequent in lipid droplets of ballooned hepatocytes. The frequency of adipophilin-positive ballooned hepatocytes was correlated to inflammation (Rs=0.72, p<0.0001), fibrosis (Rs=0.46, p=0.005), NAS (Rs=0.47, p=0.004) and oxPC-positive ballooned hepatocytes (Rs=0.35, p=0.033). CONCLUSIONS: Expression patterns of perilipin and adipophilin in NASH livers varied with the size of lipid droplets. In partiew or, adipophilin expression in ballooned hepatocytes was closely associated with oxidative damage.

Has natural selection in human populations produced two types of metabolic syndrome (with and without fatty liver)?

Fatty liver is closely related to the development of the insulin resistance syndrome that largely results from abnormal insulin signaling in three major organs: (i) skeletal muscle in which insulin sensitivity depends on fat content and metabolic activity (exercise); (ii) adipose tissue, which serves as a reservoir of energy in the form of triglycerides; and (iii) the liver, which variably serves as a source or storage site of carbohydrates and lipids. In many respects, the fatty liver resembles a mixture of brown adipose tissue (microvesicular steatosis) and white adipose tissue (macrovesicular steatosis) including the stages of fatty droplet accumulation, and the expression of uncoupling proteins and perilipin-like substances. Furthermore, the development of an inflammatory infiltrate and the increased production of cytokines as occurs in adipose tissue, suggest that the liver in some individuals serves as an extension of adipose tissue. Moreover, current evidence indicates that these morphological changes represent altered gene expression similar to that of adipocytes. However, fatty liver does not appear to be a uniform feature of the metabolic syndrome and there is substantial variation in humans in the development of fatty liver independent of insulin resistance. In this regard, the variable development of fatty liver in Palmipedes (migratory fowl) and its close relationship to skeletal muscle utilization of fatty acids, lipoprotein metabolism and thermoregulation are instructive. The predilection to non-alcoholic fatty liver disease among some varieties of Palmipedes suggests that the development of fatty liver represents an adaptive process, closely integrated with skeletal muscle fat utilization and adipose tissue distribution, and facilitates survival in a very cold, resource-scarce environment. Variation in human populations with metabolic syndrome likewise suggests that the trait evolved in populations exposed in ancient times to different environmental challenges and, because the liver plays a central role in lipid metabolism, the presence or absence of fatty liver is likely to be integrated with insulin sensitivity in other target organs and with lipoprotein metabolism.

HCV infection of the transplanted liver: changing CD81 and HVR1 variants immediately after liver transplantation.

The second envelope protein at hypervariable region 1 (HVR1) has been implicated in contributing to hepatitis C virus (HCV)-host cell interactions and CD81 (a multifunctional protein) has been demonstrated to act as a cell surface receptor for HCV and may interact directly with HVR1. The purpose of the current study was to determine if certain HVR1 quasispecies variants more effectively associate with and infect allografts after liver transplantation than other HVR1 variants and whether CD81 receptor expression changes after transplantation. Blood and allograft samples were obtained from the peritransplant period in seven patients. Clones of RT-PCR product were directly sequenced to identify HVR1 quasispecies variants. Explanted liver and serial allograft biopsies in recipients with HCV were examined by immunohistochemistry (IHC) for CD81 expression. Examination of HVR1 sequences demonstrated that only a fraction of the quasispecies variants recovered from each patient's blood sampled immediately prior to transplantation associated with and infected the allografts. Genetic diversity at HVR1 decreased with reperfusion but did not significantly decrease with infection. Expression of CD81 varied during the immediate post-transplant period. In conclusion, HVR1 quasispecies variants differentially associate with, and infect allografts, after liver transplantation. Additionally, allografts express variable amounts of CD81 after transplantation.

E2 quasispecies specificity of hepatitis C virus association with allografts immediately after liver transplantation.

It is unknown whether all hepatitis C virus (HCV) quasispecies variants found within patient serum have equal capacity to associate with the liver after transplantation; however, in vitro models of HCV infection suggest that variations in the hypervariable region 1 (HVR1) of the second envelope protein (E2) may be important in infectivity. The hypothesis of the current study is that the two hypervariable regions (HVR1 and HVR2) within E2 are important in the initial virus-liver interaction, and, therefore, certain HCV quasispecies variants will be isolated from the liver after reperfusion. In 8 patients with end-stage liver disease secondary to HCV infection, HCV envelope quasispecies were determined from intraoperative serum samples obtained before the anhepatic phase of transplantation and from liver biopsies 1.5 to 2.5 hours after the transplanted liver was perfused. Explanted (native) liver biopsies were taken as a control. Sequence analysis was performed on clones of specific HCV reverse transcriptase-polymerase chain reaction products spanning HVR1 and HVR2 of the E2 protein. HVR1 was more variable than HVR2 for all samples. Quasispecies isolated from postperfusion liver differed more from serum than did explanted liver quasispecies at HVR1 (P = 0.03) but not at HVR2 (P = 0.2). Comparison of HVR1 sequences from postperfusion liver versus serum revealed significantly less HVR1 genetic complexity and diversity (P = 0.02 and P = 0.04, respectively). Immediately after transplantation but before actual infection, liver allografts select out from the infecting serum inoculum a less heterogeneous, more closely related population of quasispecies variants.

The zonal distribution of megamitochondria with crystalline inclusions in nonalcoholic steatohepatitis.

Megamitochondria with crystalline inclusions (MMC) have been previously described in nonalcoholic fatty liver; however, their distribution within hepatic zones is unknown. We sought to determine this distribution from the core liver biopsy specimens of 31 patients: 8 males and 23 females, age range 21 to 72 years. Twenty-nine showed evidence of nonalcoholic steatohepatitis (NASH) on biopsy with steatosis, inflammation, varying degree of fibrosis, ballooned hepatocytes, and Mallory hyaline, and two patients had cryptogenic cirrhosis thought to represent "burned out" NASH. Identified by transmission electron microscopy, the abundance of MMC was compared between low-stage (fibrosis stages 1 and 2) and high-stage (fibrosis stages 3 and 4) groups and between zones with or without difference in fibrosis stage. Regardless of stage, the MMC were distributed equally in all zones and were abundant similarly in low- and high-stage groups. This abundance did not correlate with the degree of oxidative stress (4-hydroxynonenal staining) or with the abundance of ballooned hepatocytes. Consistent with age as a risk factor for more severe disease, the median age for the low-stage group was significantly lower than that of the high-stage group (P =.003). In conclusion, in NASH, the MMC seem to be distributed randomly among zones and without variation in abundance, regardless of the fibrosis stage. The exact function of these structures remains to be defined. In this study, their presence did not seem to correlate with the light microscopic injury pattern represented by ballooned hepatocytes or degree of oxidative stress defined by immunostaining for 4-hydroxynonenal.