Formalin-fixed Paraffin-embedded Liver Tissue Research Articles

Association between the Expression Patterns of Hepatitis B Surface Antigen and Hepatitis B Core Antigen with Clinicopathological Parameters and Antiviral Therapy in Liver Biopsies Obtained from Chronically Infected Hepatitis B Positive Omani Patients

Background: Hepatitis B virus (HBV) infection is considered a major global health problem. The main objectives of the current study is to determine the patterns of expression of the hepatitis B surface antigen (HBsAg) and HBV core antigen (HBcAg) in liver tissue samples obtained from hepatitis B virus-infected Omani patients and to associate between the pattern of the expression of HBsAg and HBcAg with the other clinical parameters and anti-viral therapy. Methods : The expression patterns of HBsAg and HBV core antigen HBcAg were determined by immunohistochemistry (IHC), in 58 formalin-fixed paraffin-embedded liver tissue biopsies obtained from chronic hepatitis B Omani patients. The association between positivity for HBV antigens with gender, age group, histological appearances, and antiviral therapy was determined. Results: IHC-positive staining of HBsAg was demonstrated in 28 patients (48.3%) patients, of whom 4 (6.9%) also showed HBcAg expression. The expression pattern of HBsAg was predominantly cytoplasmic and was seen in 25 (89.3%) of patients, whereas expression of HBcAg was nuclear in 3 (75%) of patients. HBsAg and HBcAg IHC positivity were more common among males than females and among those aged 39–58 years (P = 0.130 and 0.569, respectively). The presence of lymphocytic infiltration in the majority of liver biopsies examined in the present study indicates that the liver damage could be attributed to immunologically mediated events, especially because HBV is a non-cytopathogenic virus. No significant statistical association was found between positivity for HBsAg/HBcAg by IHC and antiviral treatment. Conclusions: Determination of the expression patterns of HBV antigens in liver biopsies obtained from chronic hepatitis B Omani patients and the association between these expression patterns with other clinical histopathological parameters and anti-viral therapy, will contribute greatly to a better understanding of the pathogenesis of HBV in this unique cohort group of infected individuals.

Abstract 15217: Intrahepatic Gene Expression Profile Differences in Patients With Fontan Associated Liver Disease

Introduction: Fontan associated liver disease (FALD) develops shortly after the Fontan palliation as the long-term consequence of chronically elevated central venous pressures. The molecular pathways underlying FALD remain largely unknown. We aimed to assess intrahepatic gene expression profile differences among these patients with advanced vs early fibrosis. Hypothesis: Advanced fibrosis in patients with Fontan circulation shows a specific gene expression profile with specific enriched pathways. Methods: This retrospective cohort study included adults with the Fontan circulation who had tissue available from at least one liver biopsy at the UCLA Adult Congenital Heart Disease Center. Clinical, laboratory, imaging and hemodynamic data prior to the liver biopsy were extracted from medical records. Patients were classified into early (F1-F2) or advanced fibrosis (F3-F4). RNA was isolated from formalin-fixed paraffin embedded liver tissue, RNA libraries were constructed using ribosomal RNA depletion method and sequencing was performed on Illumina Novaseq 6000. Differential gene expression and gene ontology analyses were carried out using DESeq2 and Metascape. Results: Seventy-seven patients were included, 53% women, 50% White and 20% Hispanic. Most patients had either a lateral tunnel (40%) or atriopulmonary (24%) Fontan. Four patients had 2 liver biopsies and 1 had hepatocellular carcinoma. Early and advanced fibrosis were present in 43 (54%) and 37 (46%) samples, respectively. Both groups had similar age, comorbidities, NYHA class, liver function tests and liver ultrasound features. Patients with advanced fibrosis had higher BNP, Fontan, mean pulmonary artery and capillary wedge pressures. Samples with advanced fibrosis had 921 up-regulated genes compared to the ones with early fibrosis. The up-regulated genes were enriched in extracellular matrix organization, regulation of cell adhesion, response to wounding, TGF-beta signaling pathway and blood vessel morphogenesis. Conclusions: Advanced fibrosis in patients with Fontan circulation exhibits up-regulated genes compared to early fibrosis, including pathways related to inflammation, congestion and angiogenesis, adding further insight into FALD pathophysiology.

Findings of Hepatic Severe Acute Respiratory Syndrome Coronavirus-2 Infection

Background & AimsLiver injury due to coronavirus disease 2019 (COVID-19) is being increasingly recognized. Abnormal liver chemistry tests of varying severities occur in a majority of patients. However, there is a dearth of accompanying liver histologic studies in these patients.MethodsThe current report details the clinical courses of 2 patients having severe COVID-19 hepatitis. Liver biopsies were analyzed under light microscopy, portions of liver tissue were hybridized with a target probe to the severe acute respiratory syndrome coronavirus-2 S gene, and small sections from formalin-fixed paraffin-embedded liver tissue were processed for electron microscopy.ResultsThe liver histology of both cases showed a mixed inflammatory infiltrate with prominent bile duct damage, endotheliitis, and many apoptotic bodies. In situ hybridization and electron microscopy suggest the intrahepatic presence of severe acute respiratory syndrome coronavirus-2, the findings of which may indicate the possibility of direct cell injury.ConclusionsOn the basis of the abundant apoptosis and severe cholangiocyte injury, these histopathologic changes suggest a direct cytopathic injury. Furthermore, some of the histopathologic changes may resemble acute cellular rejection occurring after liver transplantation. These 2 cases demonstrate that severe COVID-19 hepatitis can occur even in the absence of significant involvement of other organs.

Occult HBV infection status and its impact on surgical outcomes in patients with curative resection for HCV-associated hepatocellular carcinoma.

We sought to clarify the prevalence of occult hepatitis B virus (HBV) infection (OBI) and to determine whether OBI affects the surgical outcomes in curatively resected Japanese patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). A total of 257 patients with HCV-related HCC who underwent curative surgical resection were enrolled. All enrolled patients were serologically negative for HBV surface antigen and positive for HCV antibody. DNA was extracted from formalin-fixed paraffin-embedded liver tissue. OBI was determined by the HBV-DNA amplification of at least two different sets of primers by TaqMan real-time polymerase chain reaction. Surgical outcomes were evaluated according to overall survival (OS), disease-specific survival (DSS), and disease-free survival (DFS). OBI was identified in 15 of the 257 (5.8%) cases. In the multivariate analyses, the factors significantly correlated with OS were BMI >25 (P=0.0416), portal vein invasion (P=0.0065), and multiple tumors (P=0.0064). The only factor significantly correlated with DSS was T-stage (P=0.0275). The factors significantly correlated with DFS were liver fibrosis (P=0.0017) and T-stage (P=0.0001). The status of OBI did not show any significant correlation with OS, DSS or DFS, but a weak association with DSS (P=0.0603) was observed. The prevalence of OBI was 5.8% in 257 cases of HCV-related HCC. Although a weak association between DSS and OBI was observed, and statistical analyses were limited by small number of OBI cases, no significant correlation between OBI and surgical outcomes was detected.

Real-Time PCR to Detect α-1 Antitrypsin S and Z Alleles in Formalin-Fixed Paraffin-Embedded Tissue.

α-1 Antitrypsin (A1AT) deficiency is an autosomal recessive genetic disease with incomplete penetrance that can cause pulmonary and liver disease. Multiple methods are available to determine A1AT genotype using peripheral blood specimens, but none are validated to detect A1AT alleles in formalin-fixed paraffin-embedded (FFPE) tissue. A real-time PCR assay was validated to detect the SERPINA1 S and Z alleles (NM_000295.4: c.863A>T, p.E288V and c.1096G>A, p.E366K, respectively) in FFPE liver tissue using allele-specific dual hybridization probes and melting curve analysis. Validation experiments were performed on genomic DNA samples (n = 11) with A1AT genotypes previously determined by orthogonal methods. The S and Z allele assays accurately genotyped all FFPE validation specimens that had a threshold cycle <32. Validation samples produced mean melting temperatures of 55.4 °C (SD = 0.30) for mutant S alleles, 48.6 °C (SD = 0.28) for non-S alleles, 61.2 °C (SD = 0.34) for mutant Z alleles, and 54.7 °C (SD = 0.19) for non-Z alleles. Samples failing to meet quality control parameters were infrequent. Poor PCR amplification because of low nucleic acid concentration in small biopsy specimens and time-dependent degradation in specimens stored for extended periods were the most common reasons for assay failure. The ability to determine A1AT genotype from archived surgical pathology specimens can facilitate research on the role of A1AT globules in liver disease.

Whole exome sequencing in the rat

BackgroundThe rat genome was sequenced in 2004 with the aim to improve human health altered by disease and environmental influences through gene discovery and animal model validation. Here, we report development and testing of a probe set for whole exome sequencing (WES) to detect sequence variants in exons and UTRs of the rat genome. Using an in-silico approach, we designed probes targeting the rat exome and compared captured mutations in cancer-related genes from four chemically induced rat tumor cell lines (C6, FAT7, DSL-6A/C1, NBTII) to validated cancer genes in the human database, Catalogue of Somatic Mutations in Cancer (COSMIC) as well as normal rat DNA. Paired, fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) liver tissue from naive rats were sequenced to confirm known dbSNP variants and identify any additional variants.ResultsInformatics analysis of available gene annotation from rat RGSC6.0/rn6 RefSeq and Ensembl transcripts provided 223,636 unique exons representing a total of 26,365 unique genes and untranslated regions. Using this annotation and the Rn6 reference genome, an in-silico probe design generated 826,878 probe sequences of which 94.2% were uniquely aligned to the rat genome without mismatches. Further informatics analysis revealed 25,249 genes (95.8%) covered by at least one probe and 23,603 genes (93.5%) had every exon covered by one or more probes. We report high performance metrics from exome sequencing of our probe set and Sanger validation of annotated, highly relevant, cancer gene mutations as cataloged in the human COSMIC database, in addition to several exonic variants in cancer-related genes.ConclusionsAn in-silico probe set was designed to enrich the rat exome from isolated DNA. The platform was tested on rat tumor cell lines and normal FF and FFPE liver tissue. The method effectively captured target exome regions in the test DNA samples with exceptional sensitivity and specificity to obtain reliable sequencing data representing variants that are likely chemically induced somatic mutations. Genomic discovery conducted by means of high throughput WES queries should benefit investigators in discovering rat genomic variants in disease etiology and in furthering human translational research.

Markers of Tissue Repair and Cellular Aging Are Increased in the Liver Tissue of Patients With HIV Infection Regardless of Presence of HCV Coinfection.

Liver disease is a leading cause of HIV-related mortality. Hepatitis C virus (HCV)–related fibrogenesis is accelerated in the setting of HIV coinfection, yet the mechanisms underlying this aggressive pathogenesis are unclear. We identified formalin-fixed paraffin-embedded liver tissue for HIV-infected patients, HCV-infected patients, HIV/HCV-coinfected patients, and controls at Duke University Medical Center. De-identified sections were stained for markers against the wound repair Hedgehog (Hh) pathway, resident T-lymphocytes, and immune activation and cellular aging. HIV infection was independently associated with Hh activation and markers of immune dysregulation in the liver tissue.

Detection of a lipid peroxidation-induced DNA adduct across liver disease stages.

Oxidative stress and chronic inflammation can increase cellular levels of reactive oxygen species and lipid peroxidation (LPO) when associated with the pathogenesis of hepatocellular carcinoma (HCC), which can develop following the progression of steatosis, fibrosis and cirrhosis. Using a monoclonal antibody for cyclic γ-hydroxy-1, N2 -propanodeoxyguanosine (γ-OHPdG), a promutagenic DNA adduct formed endogenously by LPO, we examined its formation across liver disease stages to understand it's potential role in HCC development. Formalin-fixed paraffin embedded (FFPE) liver tissue samples from 49 patients representing normal, steatosis, fibrosis, cirrhosis and HCC were stained for γ-OHPdG and 8-hydroxydeoxyguanosine (8-oxo-dG), an oxidative damage biomarker. Quantification of immunohistochemical (IHC) staining was performed using histological scoring of intensity and distribution. Using primary human hepatocytes (HH) and a stellate cell (SC) co-culture, immunocytochemical staining of γ-OHPdG and Nile Red was performed to determine if the formation of γ-OHPdG was consistent between the clinical sample disease stages and the in vitro steatotic and fibrotic conditions. γ-OHPdG levels varied significantly between the stages of normal and steatosis, steatosis and fibrosis, and steatosis and cirrhosis (P≤0.005). There was a trend, although not significant, of increased levels of γ-OHPdG in HCC compared to the other groups. A strong correlation was observed (Pearson's, R2 =0.85) between levels of γ-OHPdG and 8-oxo-dG across the disease spectrum. The increase of γ-OHPdG in steatosis and decrease in fibrosis was a pattern confirmed in an in vitro model using primary HH co-cultured with human SCs. γ-OHPdG was detected in FFPE liver tissues of patients with different stages of liver disease and in vitro studies, demonstrating that its formation is consistent with LPO in early stages of liver disease and suggesting that it may be a source of mutagenic DNA damage in liver disease progression.

Hepatitis e virus detection in liver tissue from patients with suspected drug-induced liver injury.

Hepatitis E virus (HEV) infection is increasingly recognized as a cause of acute hepatitis in the industrialized world. We aimed to determine the frequency of acute HEV infection in cases of suspected drug-induced liver injury (DILI), mainly a diagnosis of exclusion. To this aim, formalin-fixed paraffin-embedded (FFPE) liver tissues of all cases routinely processed in our institute during a 2 1/2 years period in which DILI was among the differential diagnoses (157 liver biopsies, 1 liver explant) were subjected to semi-nested RT-PCR for the detection of HEV RNA. Histopathology was re-evaluated on all cases tested positive. HEV RNA was detectable in 3 of 158 cases (2%) tested, comprising autochthonic as well as travel-related infections with genotypes 1, 3, and 4 each found once, respectively. Histopathologic findings comprised one case with subtotal hepatic necrosis and two cases of acute (cholestatic) hepatitis not distinguishable from acute hepatitis of other etiology. Thus, the overall frequency of acute HEV infection as determined by detection of HEV RNA in liver tissue is substantially increased in patients with suspected DILI compared to the healthy population, emphasizing the need to actively look for HEV infection in cases of suspected DILI. Molecular testing for HEV RNA in routinely processed FFPE liver tissues can be applied to cases with undetermined HEV status.

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue.

BackgroundThe growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking.Experimental designDT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity.ResultsDT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility.ConclusionsThese results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.

Elevated miR-33a and miR-224 in steatotic chronic hepatitis C liver biopsies.

To assess the expression of selected microRNAs (miRNA) in hepatitis C, steatotic hepatitis C, noninfected steatotic and normal liver tissues. The relative expression levels of miR-21, miR-33a, miR-96, miR-122, miR-125b, miR-221 and miR-224 were determined in 76 RNA samples isolated from 18 non-steatotic and 28 steatotic chronic hepatitis C (CHC and CHC-Steatosis, respectively) cases, 18 non-infected, steatotic liver biopsies of metabolic origin (Steatosis) and 12 normal formalin-fixed paraffin-embedded liver tissues using TaqMan MicroRNA Assays. All CHC biopsy samples were obtained prior to initiating therapy. Patients' serum biochemical values, which included glucose, triglyceride, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl-transferase (GGT), alkaline phosphatase (AP), were obtained and correlated with relative miRNA expression. When compared with control non-infected liver samples, miR-122 and miR-221 levels were reduced in CHC-Steatosis (P < 0.03) and in CHC, CHC-Steatosis and Steatosis (P < 0.01). Alternatively, the expression of miR-33a and miR-224 were elevated in CHC-Steatosis and Steatosis in comparison to control tissue (P < 0.01). The levels of miR-33a and miR-224 in CHC-Steatosis (P < 0.02) and miR-224 in Steatosis (P < 0.001) were increased in comparison to CHC samples. By contrast, the expression of miR-21 did not differ statistically between diseased and normal liver samples. Levels of miR-33a correlated negatively with serum AST and AP levels in Steatosis as well as with necroinflammatory grade in CHC, whereas miR-21 correlated positively with AST in Steatosis and displayed negative correlation with triglyceride level in CHC-Steatosis. In contrast, miRNA levels were not correlated with ALT, GGT, cholesterol levels or fibrosis stage. Differences in miRNA expression were observed between CHC and steatotic CHC, CHC and steatotic liver, but not between steatotic CHC and steatotic liver of metabolic origin.

Molecular Diagnosis of Polycystic Echinococcosis Due to Echinococcus vogeli in a Paraguayan Immigrant in Argentina

Polycystic echinococcosis due to Echinococcus vogeli is a rare parasitic infection that occurs in rural areas of Central and South America. Only molecular identification performed on formalin-fixed paraffin-embedded liver tissue samples gave an unequivocal diagnosis of this disease in a Paraguayan immigrant in Argentina.

Mo1781 Molecular Detection of Helicobacter pylori in Formalin-Fixed Paraffin-Embedded (FFPE) Liver Tissue From Patients With Hepatocellular Carcinoma: Comparison of Five Methods to Extract Genomic DNA

hepatocellular carcinoma represents one of the most common human cancers in the world, the aim of this study was to investigate the presence of H. pylori DNA in the FFPE liver from Brazilian patients with HCC. For this purpose, paraffin sections of 38 liver biopsies were used and genomic DNA was extracted using phenol/chloroform method. After that, polymerase chain reaction (PCR) analysis was carried out using H. pylori specific 16S rRNA primers and PCR products of positive samples were further identified by DNA sequencing. Results showed that 14 of the 38 samples (36,8%) amplified the 16S rRNA H. pylori gene. The nucleotide sequence of the 16S rRNA amplicons demonstrated 98% similarity to H. pylori. Considering the H. pylori positive patients, in relation to viral infection, 9 of the 14 (64%) subjects were infected by hepatitis C virus (HCV), 2 (14%) were infected by hepatitis B virus (HBV); 1 (7%) presented coinfection with HCV and HBV and 2 (15%) patients had HCC without viral infection. These data confirm the identification of H. pylori in FFPE liver tissue of Brazilian patients with hepatocellular carcinoma. Additionally, patients infected with H. pylori were most frequently infected by HCV. Further studies will be performed in these samples to detect other H. pylori molecular markers such as cagA, vacA and ureA genes and its association with viral status of patients with HCC. This study was supported by grants from FAPESP ( 2009/09889-5) and FAEPEX (10111)

Limited yield of diagnoses of intrahepatic infectious causes of canine granulomatous hepatitis from archival liver tissue

Canine granulomatous hepatitis is an uncommon morphologic diagnosis that has been associated with a variety of diseases, including a number of systemic infectious etiologies. Formalin-fixed, paraffin-embedded (FFPE) tissues are typically the only source of liver tissue remaining for additional testing for the presence of infectious disease within granulomas. It is unclear if the more common infectious culprits of granulomatous hepatitis can be identified from such specimens. The aim of the current study was to retrospectively investigate archival FFPE liver tissue from dogs with granulomatous hepatitis for the presence of infectious agents. Semiquantitative analysis of copper accumulation in liver specimens was also performed. Medical records were examined for recorded evidence of systemic infectious disease diagnosis. Formalin-fixed, paraffin-embedded liver was prospectively evaluated for infectious agents via differential staining techniques (n = 13), eubacterial fluorescent in situ hybridization (n = 11), and Bartonella polymerase chain reaction assays (n = 15). An infectious cause of granulomatous hepatitis was not identified within liver tissue from any dog using these diagnostic methodologies. Six out of 25 (24%) dogs were diagnosed with concurrent systemic or localized bacterial infections at the time of presentation. Nine out of 17 (53%) dogs had excessive hepatic copper accumulation when evaluated by a semiquantitative histologic grading scheme or quantitative copper analysis. As definitive infectious causes of granulomatous hepatitis were not identified within archival liver biopsy samples, it was concluded that investigation of infectious etiologies within FFPE liver specimens using these diagnostic approaches may be of low yield.

Reply to the Sannier et al. letter entitled “MDR3 immunostaining on frozen liver biopsy is not a sensitive diagnosis tool for the detection of heterozygous MDR3/ABCB4 gene mutation”

Sir or Madam, We read with great interest the letter by Sannier et al. The data presented are consistent with our publication [1]. The authors studied a new series of adult patients with unexplained cholestasis and heterozygous MDR3/ABCB4 mutations. In five cases, a MDR3 immunostaining was performed on liver frozen sections and showed an intense canalicular staining similar to a normal staining. These results are in contradiction with their initial report that showed a faint MDR3 immunostaining in four patients with heterozygous MDR3/ABCB4 mutations [2]. This discrepancy may be explained by technical hazards or by the type of MDR3/ABCB4 mutations (indeed, the mutations were different in the two series). It could be interesting to perform MDR3 immunostainings on the formalin-fixed paraffin-embedded liver tissues in these two series that would enable a comparison with our published data which were obtained with this technique. To conclude, it seems that MDR3/ABCB4 heterozygous mutation diagnosis requires gene sequencing.

Quantitation of HBV covalently closed circular DNA in micro formalin fixed paraffin-embedded liver tissue using rolling circle amplification in combination with real-time PCR

Background The study aimed to develop an effective method to quantitate HBV covalently closed circular DNA (cccDNA) using small section of formalin fixed paraffin-embedded (FFPE) liver biopsy. Methods Plasmid-safe ATP-dependent DNase (PSAD)-treated samples were subjected to rolling circle amplification (RCA) prior to real-time PCR mediated by cccDNA-selective primers. Human beta-actin gene was used as a reference control. Results Compared to the classical method, i.e., PSAD digestion + real-time PCR, introduction of RCA increased the lower limit of detection for about 2 logs with good inter- and intra-assay reproducibility. HBV cccDNA was detected in 91.5% (119/130) of the FFPE samples. The cccDNA levels (copy/cell) between FFPE liver tissues and fresh frozen counterpart tissues were comparable. The median of cccDNA level in HBeAg-positive patients was higher than that in HBeAg-negative ones (52.60 vs. 31.25 copies/cell, P < 0.01). Intrahepatic cccDNA level was positively correlated with intrahepatic HBV total DNA level, but not obviously correlated with serum HBV DNA or alanine aminotransferase levels. Conclusions The method could sensitively and specifically quantitate intrahepatic HBV cccDNA in micro FFPE liver biopsy tissue for evaluation of HBV replication status in the liver.

Investigation of Primary Cilia in the Pathogenesis of Biliary Atresia

Biliary atresia (BA) is an obliterative cholangiopathy presenting in the newborn period that leads to biliary cirrhosis and the need for liver transplantation in infancy unless treated early with a palliative Kasai portoenterostomy. BA is the most frequent indication for liver transplantation in children, but the pathogenesis remains unknown (1). Abnormalities of primary cilia are the cause of syndromes such as autosomal recessive polycystic kidney disease, in which renal cyst formation is associated with liver disease (2). The liver disease in ciliopathies is a ductal plate malformation leading either to congenital hepatic fibrosis or hepatic cyst development (3). In 10% to 20% of cases with BA (syndromic BA), there is an association with other specific developmental defects, including a laterality abnormality (situs inversus). Laterality is determined by primary cilia, and genes affecting laterality are involved in the organisation of cilial microtubules within the embryonal central node (4). In the present study we investigated children with BA who developed renal cysts to increase our understanding of the pathological process. We evaluated the effect of liver transplantation on cyst formation, assessed the role of primary cilia using fibrocystin as a cilial marker and looked for PKHD1 mutations in this group, and finally determined the function of their motile respiratory cilia.

Study of Viral Infections in Infants with Biliary Atresia

Viral infections may have an important role in the pathogenesis of biliary atresia, and related clinical outcomes. In this research for determination of the possible role of HBV, HCV, HCMV, adenovirus, and BK virus infections in biliary atresia related clinical complications, the molecular and antigenic prevalence of these viral agents were studied. In this retrospective study, 34 formalin fixed paraffin embedded (FFPE) biopsy and autopsy liver tissue samples of neonates with biliary atresia were evaluated. The molecular prevalence of these viral infections was assayed by different PCR and RT-PCR methods. The antigenic prevalence of HBV, HCV, and HCMV infections was also studied in these liver tissue samples by immunohistochemistry (IHC) method. HBV, HCV, and adenovirus genomes were detected in 9%, 6%, and 6% of liver autopsy and biopsy tissues of infants with biliary atresia, respectively. HBV and HCV co-infection was confirmed in 6% of FFPE samples. The genome of other investigated viruses was not detected in FFPE liver tissues. Detection of viral infection in FFPE liver tissue samples of newborns with biliary atresia, suggests the need for complete studies for the determination of accurate role of these viral infections in pathogenesis of biliary atresia.

Seasonal Outbreaks of Tyzzer's Disease in Malagasy Giant Jumping Rats ( Hypogeomys antimena) in the Riga Zoo

RESULTS: Acute hepatic necrosis was detected in 3/4 rats examined from 2004 outbreak and in both rats from 2009 outbreak. Large, slender bacilli, typical of Clostridium piliforme were seen in Warthin-Starry silver stained sections. In 2009 cases C. piliforme DNA was found in formalin-fixed paraffin-embedded liver tissues by PCR. Sequencing of conserved region of C. piliforme showed that the strain was 99% homologous to mouse isolate. These findings support Tyzzer‟s disease as the cause for both outbreaks.

Detection of hepatitis B virus covalently closed circular DNA in paraffin-embedded and cryo-preserved liver biopsies of chronic hepatitis B patients

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) may become an important predictor for treatment outcome or long-term follow-up. To detect cccDNA in formalin-fixed, paraffin embedded (FFPE) and to compare with cryo-preserved liver tissue. Biopsies of 56 chronic hepatitis B patients were collected. Cryo-preserved and FFPE liver biopsies were available from 37 out of 56 patients. Paraffin was extracted with 1 ml xylene, followed by 100% alcohol and acetone. For the detection of cccDNA, selective primers were used. For quantification of hepatocytes a commercial Taqman beta-actin control kit was used. The cccDNA was detected in 80% of FFPE and in 100% of cryo-preserved liver specimens. Recovery of hepatocytes and cccDNA was approximately a 100-fold lower in FFPE liver tissue, but intrahepatic cccDNA levels were comparable. In FFPE and cryo-preserved liver tissue, intrahepatic cccDNA levels correlated strongly with HBV DNA, hepatitis B e antigen (HbeAg), and plasma cccDNA levels. HbeAg positive chronic hepatitis B patients had significantly higher intrahepatic cccDNA levels compared with HBeAg negative patients (P<0.05). In HBeAg positive patients, no difference in intrahepatic cccDNA levels were seen between patients with active (histological activity index score>3; HBV DNA>20 000 IU/ml) and inactive hepatitis (histological activity index score</=3). In HBeAg negative chronic hepatitis B patients, intrahepatic cccDNA levels were significantly higher in patients with active hepatitis (P=0.004 and 0.001). Recovery of hepatocytes and cccDNA in FFPE tissue was lower, but intrahepatic cccDNA in FFPE biopsies were comparable with cryo-preserved liver tissue. Therefore, FFPE liver tissue is an attractive alternative for cccDNA analysis when cryo-preserved tissue is not available.