scholarly journals Equine Parvovirus-Hepatitis Screening in Horses and Donkeys with Histopathologic Liver Abnormalities

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1599
Author(s):  
Verena Zehetner ◽  
Jessika-M.V. Cavalleri ◽  
Andrea Klang ◽  
Martin Hofer ◽  
Irina Preining ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Normal Liver ◽  
Digital Pcr ◽  
Hepatic Necrosis ◽  
Neoplastic Disease ◽  
Viral Loads ◽  
Hepatic Diseases ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact

There is strong evidence that equine parvovirus-hepatitis (EqPV-H) is associated with the onset of Theiler’s disease, an acute hepatic necrosis, in horses. However, the impact of this virus on other hepatopathies remains unknown. The objective of this retrospective study was to evaluate the prevalence and quantify the viral loads of EqPV-H in formalin-fixed, paraffin-embedded equine and donkey livers with various histopathologic abnormalities. The pathologies included cirrhosis, circulatory disorders of the liver, toxic and metabolic hepatic diseases as well as neoplastic and inflammatory diseases (n = 84). Eight normal liver samples were included for comparison as controls. EqPV-H DNA was qualitatively and quantitatively measured by real-time PCR and digital PCR, respectively. The virus was detected in two livers originating from horses diagnosed with abdominal neoplasia and liver metastasis (loads of 5 × 103 and 9.5 × 103 genome equivalents per million cells). The amount of viral nucleic acids measured indicates chronic infection or persistence of EqPV-H, which might have been facilitated by the neoplastic disease. In summary, this study did not provide evidence for EqPV-H being involved in hepatopathies other than Theiler’s disease.

The Progenitor Cell Compartment in the Feline Liver: An (Immuno)Histochemical Investigation

2009 ◽  
Vol 46 (4) ◽  
pp. 614-621 ◽  
Author(s):  
J. Ijzer ◽  
J. R. Kisjes ◽  
L. C. Penning ◽  
J. Rothuizen ◽  
T. S. G. A. M Van Den Inch
Keyword(s):  
Progenitor Cells ◽  
Progenitor Cell ◽  
Normal Liver ◽  
Hepatic Progenitor Cells ◽  
Ductular Reaction ◽  
Hepatic Diseases ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Mitotic Figures ◽  
Progenitor Compartment

The hepatic progenitor compartment is of vital importance in liver regeneration when hepatocellular replication is impaired, as it occurs in acute fulminant hepatitis or severe liver fibrosis. It consists of resident progenitor cells in the normal liver, and ductular reaction and intermediate hepatobiliary cells in diseased livers. An histologic and immunohistochemical study was conducted to demonstrate putative hepatic progenitor cells in the normal liver (n = 5) and in a range of hepatic diseases (n = 13) in the cat. Formalin-fixed, paraffin-embedded specimens were stained with HE, the van Gieson stain, and the reticulin stain according to Gordon and Sweet, and immunohistochemically stained for cytokeratin-7 (CK7), human hepatocyte marker 1 (Heparl), and multidrug resistance-binding protein-2/ATP binding cassette C2 (MRP2). The normal feline liver contains a liver progenitor cell morphologically similar to humans and dogs, which resides in the canal of Hering. In acute and chronic feline liver diseases a ductular reaction is present, whether in the parenchyma or in a portal or septal location. The putative progenitor cells could easily be demonstrated by staining for CK7, whereas they were generally negative for Heparl and MRP2. In a parenchymal ductular reaction mitotic figures and cells with an intermediate hepatobiliary phenotype could be demonstrated. This is the first account of hepatic progenitor cells in feline liver.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

scholarly journals Droplet Digital PCR for Mutation Detection in Formalin-Fixed, Paraffin-Embedded Melanoma Tissues

2018 ◽  
Vol 20 (2) ◽  
pp. 240-252 ◽  
Author(s):  
Ashleigh C. McEvoy ◽  
Benjamin A. Wood ◽  
Nima M. Ardakani ◽  
Michelle R. Pereira ◽  
Robert Pearce ◽  
...  
Keyword(s):  
Mutation Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals Single-day HER2neu amplification assessment using chip-based digital PCR in formalin-fixed paraffin-embedded breast carcinoma tissue

2018 ◽  
Vol Volume 10 ◽  
pp. 121-129
Author(s):  
Parth Shah ◽  
Shiva Murarka ◽  
Anupam Joshi ◽  
Bhavana Mehta ◽  
Vipal Parmar ◽  
...  
Keyword(s):  
Breast Carcinoma ◽  
Digital Pcr ◽  
Carcinoma Tissue ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Quantitative immunohistochemical (IHC) assessment of ataxia-telangiectasia mutated (ATM) in estrogen receptor (ER) negative early breast cancer (BC).

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e21083-e21083
Author(s):  
Patricia Tang ◽  
Alexander C. Klimowicz ◽  
Gregory Russell Pond ◽  
Daniel Yick Chin Heng ◽  
Marc A. Webster ◽  
...  
Keyword(s):  
Cellular Response ◽  
Cox Regression ◽  
Stage I ◽  
Ataxia Telangiectasia Mutated ◽  
Expression Ratio ◽  
Regression Methods ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Quantitative Fluorescence

e21083 Background: ATM plays a key role in the cellular response to DNA damage and germline mutations are associated with a predisposition to BC. We evaluated the impact of ATM expression on outcomes in Stage I-III ER negative BC patients (pts). Methods: A tissue microarray was constructed from formalin-fixed paraffin-embedded specimens of ER negative Stage I-III BC pts from the Tom Baker Cancer Centre. ATM expression was assessed by quantitative fluorescence IHC using a rabbit anti-ATM monoclonal antibody (Epitomics) and the HistoRx AQUA platform. ATM expression index (EI) was calculated by the tumor cell to stroma expression ratio. Prognostic ability of ATM EI was explored univariately, and multivariately after adjusting for clinicopathological factors selected using algorithmic methods, with Cox regression methods. Results: Of 126 eligible pts treated from 1999-2004, 44.4% were HER2-neu + and 52.4% were triple negative. ATM EI was normalized using a logarithmic transformation. Univariately, higher ATM EI was associated with worse outcomes (Table). Multivariately, higher ATM EI remained significantly prognostic for recurrence-free survival (RFS) (HR=5.46, p=0.046), local RFS (HR=5.56, p=0.052), distant RFS (HR=4.38, p=0.080), but not overall survival (p=0.48). Conclusions: High ATM EI is associated with worse RFS in ER negative BC in this hypothesis-generating study. Validation of this finding is currently ongoing. [Table: see text]

scholarly journals Accuracy and Clinical Relevance of Intra-Tumoral Fusobacterium nucleatum Detection in Formalin-Fixed Paraffin-Embedded (FFPE) Tissue by Droplet Digital PCR (ddPCR) in Colorectal Cancer

Diagnostics ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 114
Author(s):  
José Guilherme Datorre ◽  
Ana Carolina de Carvalho ◽  
Mariana Bisarro dos Reis ◽  
Monise dos Reis ◽  
Marcus Matsushita ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Clinical Relevance ◽  
Fusobacterium Nucleatum ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Detection Accuracy ◽  
Assay Optimization ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

The use of droplet digital PCR (ddPCR) to identify and quantify low-abundance targets is a significant advantage for accurately detecting potentially oncogenic bacteria. Fusobacterium nucleatum (Fn) is implicated in colorectal cancer (CRC) tumorigenesis and is becoming an important prognostic biomarker. We evaluated the detection accuracy and clinical relevance of Fn DNA by ddPCR in a molecularly characterized, formalin-fixed, paraffin-embedded (FFPE) CRC cohort previously analyzed by qPCR for Fn levels. Following a ddPCR assay optimization and an analytical evaluation, Fn DNA were measured in 139 CRC FFPE cases. The measures of accuracy for Fn status compared to the prior results generated by qPCR and the association with clinicopathological and molecular patients’ features were also evaluated. The ddPCR-based Fn assay was sensitive and specific to positive controls. Fn DNA were detected in 20.1% of cases and further classified as Fn-high and Fn-low/negative, according to the median amount of Fn DNA that were detected in all cases and associated with the patient’s worst prognosis. There was a low agreement between the Fn status determined by ddPCR and qPCR (Cohen’s Kappa = 0.210). Our findings show that ddPCR can detect and quantify Fn in FFPE tumor tissues and highlights its clinical relevance in Fn detection in a routine CRC setting.

scholarly journals Automated PCR detection of BRAF mutations in colorectal adenocarcinoma: a diagnostic test accuracy study

2015 ◽  
Vol 69 (5) ◽  
pp. 398-402 ◽  
Author(s):  
Richard Colling ◽  
Lai Mun Wang ◽  
Elizabeth Soilleux
Keyword(s):  
Digital Pcr ◽  
Turnaround Time ◽  
Test Accuracy ◽  
Braf Mutations ◽  
System A ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Accuracy Study ◽  
Automated Platform ◽  
Formalin Fixed

BackgroundTesting for BRAF mutations in colorectal carcinoma (CRC) is important in the screening pathway for Lynch syndrome and is of prognostic value to guide management. This is a diagnostic accuracy study of the Idylla system, a novel and automated alternative PCR system.Methods100 consecutive formalin-fixed, paraffin-embedded CRC resection cases were tested for BRAF mutations using the Idylla automated platform and compared with standard (Cobas) PCR.ResultsThe sensitivity of the Idylla BRAF test was 100% and the specificity was 96%. Only one discordant Idylla positive/standard PCR negative result occurred and on Droplet Digital PCR demonstrated a mutation not identified by traditional PCR in this case.ConclusionThis study has validated the Idylla system for BRAF testing in CRC and demonstrated a possibly greater sensitivity, in addition to cost effectiveness and shorter turnaround time, when compared with standard PCR.

scholarly journals Next Generation Digital PCR Measurement of Hepatitis B Virus Copy Number in Formalin-Fixed Paraffin-Embedded Hepatocellular Carcinoma Tissue

2015 ◽  
Vol 61 (1) ◽  
pp. 290-296 ◽  
Author(s):  
Jing-Tao Huang ◽  
Ying-Juan Liu ◽  
Jin Wang ◽  
Zhi-Gao Xu ◽  
Ying Yang ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Copy Number ◽  
Digital Pcr ◽  
Hbv Infection ◽  
Copy Numbers ◽  
Formalin Fixed Paraffin ◽  
B Virus ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Abstract BACKGROUND Hepatocellular carcinoma (HCC) is strongly associated with hepatitis B virus (HBV) infection. False-negative results are common in routine serological tests and quantitative real-time PCR because of HBV surface antigen (HBsAg) variation and low HBV copy number. Droplet digital PCR (ddPCR), a next generation digital PCR, is a novel, sensitive, and specific platform that can be used to improve HBV detection. METHODS A total of 131 HCC cases with different tumor stages and clinical features were initially classified with a serological test as HBsAg positive (n = 107) or negative (n = 24) for HBV infection. Next, DNA templates were prepared from the corresponding formalin-fixed paraffin-embedded (FFPE) tissues to determine HBV copy number by ddPCR. RESULTS HBV copy numbers, successfully determined for all clinical FFPE tissues (n = 131), ranged from 1.1 to 175.5 copies/μL according to ddPCR. The copy numbers of HBV were positively correlated with tumor-nodes-metastasis (P = 0.008) and Barcelona-Clinic Liver Cancer (P = 0.045) classification. Moreover, serum cholinesterase correlated with hepatitis B viral load (P = 0.006). CONCLUSIONS HBV infection is a key factor that influences tumorigenesis in HCC by regulating tumor occurrence and development. ddPCR improves the analytical sensitivity and specificity of measurements in nucleic acids at a single-molecule level and is suitable for HBV detection.

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