Residual Humidity in Paraffin-Embedded Tissue Reduces Nucleic Acid Stability.

Molecular diagnostics in healthcare relies increasingly on genomic and transcriptomic methodologies and requires appropriate tissue specimens from which nucleic acids (NA) of sufficiently high quality can be obtained. Besides the duration of ischemia and fixation type, NA quality depends on a variety of other pre-analytical parameters, such as storage conditions and duration. It has been discussed that the improper dehydration of tissue during processing influences the quality of NAs and the shelf life of fixed tissue. Here, we report on establishing a method for determining the amount of residual water in fixed, paraffin-embedded tissue (fixed by neutral buffered formalin or a non-crosslinking fixative) and its correlation to the performance of NAs in quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The amount of residual water depended primarily on the fixative type and the dehydration protocol and, to a lesser extent, on storage conditions and time. Moreover, we found that these parameters were associated with the qRT-PCR performance of extracted NAs. Besides the cross-linking of NAs and the modification of nucleobases by formalin, the hydrolysis of NAs by residual water was found to contribute to reduced qRT-PCR performance. The negative effects of residual water on NA stability are not only important for the design and interpretation of research but must also be taken into account in clinical diagnostics where the reanalysis of archived tissue from a primary tumor may be required (e.g., after disease recurrence). We conclude that improving the shelf life of fixed tissue requires meticulous dehydration and dry storage to minimize the degradative influence of residual water on NAs.

Explainability and causability for artificial intelligence-supported medical image analysis in the context of the European In Vitro Diagnostic Regulation.

Artificial Intelligence (AI) for the biomedical domain is gaining significant interest and holds considerable potential for the future of healthcare, particularly also in the context of in vitro diagnostics. The European In Vitro Diagnostic Medical Device Regulation (IVDR) explicitly includes software in its requirements. This poses major challenges for In Vitro Diagnostic devices (IVDs) that involve Machine Learning (ML) algorithms for data analysis and decision support. This can increase the difficulty of applying some of the most successful ML and Deep Learning (DL) methods to the biomedical domain, just by missing the required explanatory components from the manufacturers. In this context, trustworthy AI has to empower biomedical professionals to take responsibility for their decision-making, which clearly raises the need for explainable AI methods. Explainable AI, such as layer-wise relevance propagation, can help in highlighting the relevant parts of inputs to, and representations in, a neural network that caused a result and visualize these relevant parts. In the same way that usability encompasses measurements for the quality of use, the concept of causability encompasses measurements for the quality of explanations produced by explainable AI methods. This paper describes both concepts and gives examples of how explainability and causability are essential in order to demonstrate scientific validity as well as analytical and clinical performance for future AI-based IVDs.

Biobanks for life sciences and personalized medicine: importance of standardization, biosafety, biosecurity, and data management.

Biological samples such as tissues, blood and other body fluids, plants or seeds, prokaryotic and eukaryotic cells or isolated biomolecules as well as associated data are the essential raw material for research and development in medicine, biotechnology and agriculture. The collection, processing, preservation, and storage of these resources, in addition to provision of access, are key activities of biobanks or biological resource centres. Biobanks have to ensure proper quality of samples and data, ethical and legal compliance as well as transparent and efficient access procedures. In this context the review places special emphasis on pre-analytical procedures and international standards, which are essential to improving analytical data reliability and reproducibility, as well as on the increasing importance of data management. These requirements of biobanks are demonstrated using the example of pathogen-containing and microbiome biobanks, and refer to needs in cancer research and development.

Pre-analytical processes in medical diagnostics: New regulatory requirements and standards.

In May 2017, the European In Vitro Diagnostic Regulation (IVDR) entered into force and will apply to in vitro diagnostics from May 26th, 2022. This will have a major impact on the in vitro diagnostics (IVD) industry as all devices falling under the scope of the IVDR will require new or re-certification. It will also affect health institutions developing and using in-house devices. The IVDR also has implications with respect to product performance validation and verification including the pre-analytics of biological samples used by IVD developers and diagnostic service providers. In parallel to the IVDR, a series of standards on pre-analytical sample processing has been published by the International Organization for Standardization (ISO) and the European Committee for Standardization (CEN). These standards describe pre-analytical requirements for various types of analyses in various types of biospecimens. They are of relevance for IVD product developers in the context of (re)certification under the IVDR and to some extent also to devices manufactured and used only within health institutions. This review highlights the background and the rational for the pre-analytical standards. It describes the procedure that leads to these standards, the major implications of the standards and the requirements on pre-analytical workflows. In addition, it discusses the relationship between the standards and the IVDR.

The impact of crosslinking and non-crosslinking fixatives on antigen retrieval and immunohistochemistry.

Pre-analytical factors can greatly influence the outcome of molecular analyses in medical diagnostics and research. This also applies to in situ staining techniques such as immunohistochemistry (IHC), where different types of tissue fixation methods lead to different modifications of proteins and thus can affect differently the detection by antibodies. For formalin-fixed paraffin-embedded (FFPE) tissue, antigen retrieval is applied in order to reverse the negative effects of formalin and re-establish immunoreactivity. Most antibodies and protocols used in IHC are optimized for FFPE tissue, but not for paraffin-embedded tissue treated with other fixatives such as non-crosslinking fixatives. We report results from systematic studies on distinct pre-analytical conditions in IHC, immunofluorescence and electron microscopy. Parameters investigated are the impact of crosslinking and non-crosslinking fixatives (comparing formalin and PAXgene Tissue fixation) on whole tissue, subcellular structures and organelles, as well as on ultrastructure. The results generated show that minor changes in antigen retrieval conditions may have a major impact on IHC results and that protocols optimized for crosslinking fixatives may not be used for other fixatives without re-validation. Key antigen retrieval parameters such as buffers with different pH and duration of microwave treatment must be tested systematically for each antibody and fixation protocol.

High Keratin 8/18 Ratio Predicts Aggressive Hepatocellular Cancer Phenotype.

BACKGROUND & AIMS: Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, liver cell ballooning, cytoplasmic aggregates termed Mallory-Denk bodies (MDBs), inflammation, and fibrosis at late stage. Disturbance of the keratin cytoskeleton and aggregation of keratins (KRTs) are essential for MDB formation. METHODS: We analyzed livers of aged Krt18-/- mice that spontaneously developed in the majority of cases SH-associated HCC independent of sex. Interestingly, the hepatic lipid profile in Krt18-/- mice, which accumulate KRT8, closely resembles human SH lipid profiles and shows that the excess of KRT8 over KRT18 determines the likelihood to develop SH-associated HCC linked with enhanced lipogenesis. RESULTS: Our analysis of the genetic profile of Krt18-/- mice with 26 human hepatoma cell lines and with data sets of >300 patients with HCC, where Krt18-/- gene signatures matched human HCC. Interestingly, a high KRT8/18 ratio is associated with an aggressive HCC phenotype. CONCLUSIONS: We can prove that intermediate filaments and their binding partners are tightly linked to hepatic lipid metabolism and to hepatocarcinogenesis. We suggest KRT8/18 ratio as a novel HCC biomarker for HCC.

Keratin 18-deficiency results in steatohepatitis and liver tumors in old mice: A model of steatohepatitis-associated liver carcinogenesis.

Backround: Steatohepatitis (SH)-associated liver carcinogenesis is an increasingly important issue in clinical medicine. SH is morphologically characterized by steatosis, hepatocyte injury, ballooning, hepatocytic cytoplasmic inclusions termed Mallory-Denk bodies (MDBs), inflammation and fibrosis. RESULTS: 17-20-months-old Krt18-/- and Krt18+/- mice in contrast to wt mice spontaneously developed liver lesions closely resembling the morphological spectrum of human SH as well as liver tumors. The pathologic alterations were more pronounced in Krt18-/- than in Krt18+/- mice. The frequency of liver tumors with male predominance was significantly higher in Krt18-/- compared to age-matched Krt18+/- and wt mice. Krt18-deficient tumors in contrast to wt animals displayed SH features and often pleomorphic morphology. aCGH analysis of tumors revealed chromosomal aberrations in Krt18-/- liver tumors, affecting loci of oncogenes and tumor suppressor genes. MATERIALS AND METHODS: Livers of 3-, 6-, 12- and 17-20-months-old aged wild type (wt), Krt18+/- and Krt18-/- (129P2/OlaHsd background) mice were analyzed by light and immunofluorescence microscopy as well as immunohistochemistry. Liver tumors arising in aged mice were analyzed by array comparative genomic hybridization (aCGH). CONCLUSIONS: Our findings show that K18 deficiency of hepatocytes leads to steatosis, increasing with age, and finally to SH. K18 deficiency and age promote liver tumor development in mice, frequently on the basis of chromosomal instability, resembling human HCC with stemness features.

Immunofluorescence and Immunohistochemical Detection of Keratins.

Reliable detection of keratins in tissues is important for investigating their physiological role and for using keratin expression as a biomarker in medical diagnostics. A particular challenge for the detection of keratins by immunofluorescence microscopy or immunohistochemistry relates to the fact that keratin intermediate filaments are obligatory heteropolymers, which may result in dissociation between RNA and protein expression levels in the event that the homeostasis of the expression of the proper keratin partners is disturbed. Furthermore, variable accessibility of epitopes on keratin polypeptides due to conformational changes may lead to false negative results. Preanalytical effects, such as warm/cold ischemia, fixation, tissue processing, and embedding may result in false negative or inappropriate reactions. An experimental design for how to systematically test preanalytical effects and to validate immunohistochemistry protocols is presented. This kind of evaluation should be performed for each antigen and antibody since the various epitopes recognized by antibodies may behave differently. In this context, one has to be aware that different cell structures may be affected or modified differently by various preanalytical procedures and may thus require different preanalytical and staining protocols.

Genetic background effects of keratin 8 and 18 in a DDC-induced hepatotoxicity and Mallory-Denk body formation mouse model.

Keratin 8 (K8) and keratin 18 (K18) form the major hepatocyte cytoskeleton. We investigated the impact of genetic loss of either K8 or K18 on liver homeostasis under toxic stress with the hypothesis that K8 and K18 exert different functions. krt8⁻/⁻ and krt18⁻/⁻ mice crossed into the same 129-ola genetic background were treated by acute and chronic administration of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). In acutely DDC-intoxicated mice, macrovesicular steatosis was more pronounced in krt8⁻/⁻ and krt18⁻/⁻ compared with wild-type (wt) animals. Mallory-Denk bodies (MDBs) appeared in krt18⁻/⁻ mice already at an early stage of intoxication in contrast to krt8⁻/⁻ mice that did not display MDB formation when fed with DDC. Keratin-deficient mice displayed significantly lower numbers of apoptotic hepatocytes than wt animals. krt8⁻/⁻, krt18⁻/⁻ and control mice displayed comparable cell proliferation rates. Chronically DDC-intoxicated krt18⁻/⁻ and wt mice showed a similarly increased degree of steatohepatitis with hepatocyte ballooning and MDB formation. In krt8⁻/⁻ mice, steatosis was less, ballooning, and MDBs were absent. krt18⁻/⁻ mice developed MDBs whereas krt8⁻/⁻ mice on the same genetic background did not, highlighting the significance of different structural properties of keratins. They are independent of the genetic background as an intrinsic factor. By contrast, toxicity effects may depend on the genetic background. krt8⁻/⁻ and krt18⁻/⁻ mice on the same genetic background show similar sensitivity to DDC intoxication and almost resemble wt animals regarding survival, degree of porphyria, liver-to-body weight ratio, serum bilirubin and liver enzyme levels. This stands in contrast to previous work where krt8⁻/⁻ and krt18⁻/⁻ mice on different genetic backgrounds were investigated.

From Mallory to Mallory-Denk bodies: what, how and why?

Frank B. Mallory described cytoplasmic hyaline inclusions in hepatocytes of patients with alcoholic hepatitis in 1911. These inclusions became known as Mallory bodies (MBs) and have since been associated with a variety of other liver diseases including non-alcoholic fatty liver disease. Helmut Denk and colleagues described the first animal model of MBs in 1975 that involves feeding mice griseofulvin. Since then, mouse models have been instrumental in helping understand the pathogenesis of MBs. Given the tremendous contributions made by Denk to the field, we propose renaming MBs as Mallory-Denk bodies (MDBs). The major constituents of MDBs include keratins 8 and 18 (K8/18), ubiquitin, and p62. The relevant proteins and cellular processes that contribute to MDB formation and accumulation include the type of chronic stress, the extent of stress-induced protein misfolding and consequent proteasome overload, a K8-greater-than-K18 ratio, transamidation of K8 and other proteins, presence of p62 and autophagy. Although it remains unclear whether MDBs serve a bystander, protective or injury promoting function, they do serve an important role as histological and potential progression markers in several liver diseases.

p62 protein is expressed in pancreatic beta cells.

p62 is a cellular protein that plays an adapter role in signal transduction pathways involved in such diverse biological functions as proliferation, differentiation, reaction to oxidative stress and immune response. Furthermore, p62 has recently been detected as a component of intracytoplasmic protein aggregates (inclusion bodies), which are hallmarks of a variety of chronic degenerative disorders, such as Parkinson's disease and Alzheimer's disease, but also of steatohepatitis. Here we report that p62 and insulin are co-expressed in a diffuse fashion in beta cells in normal human pancreas as well as in primary chronic pancreatitis and in normal pancreas from mouse and swine. In contrast, p62 protein is absent from, or only focally and very weakly expressed in, insulinomas, glucagonomas or non-functioning pancreatic neuroendocrine tumours or carcinomas that express insulin or other pancreatic as well as extrapancreatic hormones. Although the biological function of p62 in beta cells is unknown, the co-expression of p62 and insulin in non-neoplastic beta cells suggests that, in the beta cell, p62 may play a role in specific insulin-related signalling. Since p62 may also be involved in pro-apototic signal transduction, the loss of p62 expression in neuroendocrine neoplasms of the pancreas may render the tumour cells less sensitive to pro-apototic signals. Further research is necessary to elucidate the role of p62 in beta cell-specific signal transduction.

Interaction of stress proteins with misfolded keratins.

Misfolded and aggregated proteins are a characteristic feature of a variety of chronic diseases. Examples include neurofibrillary tangles in Alzheimer disease, Lewy bodies in Parkinson disease and Mallory bodies (MBs) in chronic liver diseases, particularly alcoholic and non-alcoholic steatohepatitis (ASH and NASH). MB formation is at least in part the result of chronic oxidative cell stress in hepatocytes and can be induced in mice by long-term intoxication with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Proteomic analysis revealed that MBs consist of ubiquitinated keratins and the stress proteins Hsp70, Hsp25, and p62. Furthermore, marked overexpression of clusterin, which shares functional properties with small heat shock proteins, was identified by gene expression profiling of DDC-treated mice livers. To investigate whether clusterin has a function in the stress response to misfolded keratins, we performed transfection studies utilizing expression constructs encoding ubiquitin, p62, Hsp27, clusterin, keratin 8, and keratin 18. Ubiquitin was found in a strong and constant association with keratin aggregates, whereas binding of p62 to keratin was variable. Hsp27 did not colocalize with keratin aggregates under these experimental conditions. In contrast, clusterin associated with misfolded keratin only if its signal peptide was deleted and its secretion inhibited. This suggests that clusterin has ability to bind misfolded proteins, including keratins but its physiological function is restricted to the extracellular space. The extracellular localization of clusterin was underlined by immunohistochemical studies in Alzheimer disease brains, where clusterin was constantly found in association with amyloid plaques; in contrast, cytoplasmic inclusions such as neurofibrillary tangles as well as MBs in ASH were negative. Furthermore, we found clusterin in association with elastic fibers in the extracellular matrix in several chronic liver diseases, including ASH and alpha1-antitrypsin deficiency, implying a possible role of clusterin in liver fibrosis.

Mallory body formation in primary biliary cirrhosis is associated with increased amounts and abnormal phosphorylation and ubiquitination of cytokeratins.

BACKGROUND/AIMS: Animal studies revealed a key role of toxic bile acids in the regulation of hepatocytic cytokeratin (CK) expression and Mallory body (MB) formation. In this study, we compared CK expression, phosphorylation, and ubiquitination in primary biliary cirrhosis (PBC), chronic hepatitis C (CHC) and control livers to determine whether bile acid-induced CK alterations are associated with cytoskeletal alterations and MB formation in a prototypic chronic cholestatic liver disease. METHODS: CK 8 and CK 18 mRNA and protein levels were investigated by reverse transcriptase-polymerase chain reaction and Western blotting. Intermediate filament (IF) cytoskeletal alterations were assessed by immunofluorescence microscopy using antibodies against CKs, CK phosphoepitopes, MBs, and ubiquitin. RESULTS: Despite unchanged mRNA levels, CK 8 and CK 18 protein levels were significantly elevated in PBC suggesting stabilization of CKs, possibly due to decreased degradation. CK-IF alterations in PBC comprised increased density with abnormal phosphorylation of the IF network of hepatocytes in acinar zone 1 and in the periphery of cirrhotic nodules. In addition, in these areas hepatocytes with diminished IF network containing MBs consisting of abnormally phosphorylated and ubiquitinated CK were observed. CONCLUSIONS: These findings support our concept that IF cytoskeletal alterations and MB formation in cholestatic liver diseases are related to bile acid-induced cell stress.

Mallory body--a disease-associated type of sequestosome.

Mallory bodies (MBs) consist of abnormal keratins, ubiquitin, heat shock proteins, and the protein p62. p62 is encoded by an immediate-early response gene that rapidly responds to a variety of extracellular signals involved in cell proliferation, differentiation, and particularly oxidative stress. It acts as an adapter in signal transduction and binds noncovalently to ubiquitin, possibly being involved in the regulation of the fate of ubiquitinated proteins by segregation (i.e., sequestosome or aggresome formation). The presence of p62 together with ubiquitinated abnormal keratins in the MB characterizes MBs as a disease-associated type of sequestosome. A detailed study on the expression of p62 and its relationship to MB formation in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-treated mouse liver is reported based on immunohistochemical, immunoblot, and Northern blot analyses. The results indicate that p62 is rapidly induced in hepatocytes of intoxicated animals preceding MB formation. As suggested by experiments with short-term DDC-treated naive mice and mice refed DDC after recovery from long-term DDC treatment (primed mice), p62 does not exert an initiating effect on MB formation but the appearance of MBs requires the presence of abnormal keratins, which associate with p62 after ubiquitination. The rapid induction of p62 and its association with MBs further support the role of oxidative stress in MB formation. In conclusion, the constant presence of p62 in MBs suggests that binding of p62 to abnormal keratins may allow hepatocytes to dispose potentially harmful proteins in a biologically inert manner.

p62 Is a common component of cytoplasmic inclusions in protein aggregation diseases.

Exposure of cells to stress, particularly oxidative stress, leads to misfolding of proteins and, if they are not refolded or degraded, to cytoplasmic protein aggregates. Protein aggregates are characteristic features of a variety of chronic toxic and degenerative diseases, such as Mallory bodies (MBs) in hepatocytes in alcoholic and non-alcoholic steatohepatitis, neurofibrillary tangles in neurons in Alzheimer's, and Lewy bodies in Parkinson's disease. Using 2D gel electrophoresis and mass spectrometry, we identified p62 as a novel MB component. p62 and cytokeratins (CKs) are major MB constituents; HSP 70, HSP 25, and ubiquitinated CKs are also present. These proteins characterize MBs as a prototype of disease-associated cytoplasmic inclusions generated by stress-induced protein misfolding. As revealed by transfection of tissue culture cells overexpressed p62 did not induce aggregation of regular CK filaments but selectively bound to misfolded and ubiquitinated CKs. The general role of p62 in the cellular response to misfolded proteins was substantiated by detection of p62 in other cytoplasmic inclusions, such as neurofibrillary tangles, Lewy bodies, Rosenthal fibers, intracytoplasmic hyaline bodies in hepatocellular carcinoma, and alpha1-antitrypsin aggregates. The presence of p62 along with other stress proteins and ubiquitin in cytoplasmic inclusions indicates deposition as aggregates as a third line of defense against misfolded proteins in addition to refolding and degradation.