**7. Functional liver tissue analysis in biopsy**

The diagnostic evaluation of liver biopsy is mostly based on panel of histochemical stains including hematoxylin-eosin [2], Masson's trichrome [3], PAS [5] and Perl's [1] stains as well as others by necessity. These visualisation techniques should be comple‐ mented by various "- omics" tools [27] to gain more data on the function of liver cells. The cytokines, inflammatory mediators, viral proteins, cell cycle proteins and apoptosis markers can be detected; metabolic pathways can be investigated as well. At present, most or proteomic and genetic studies are carried out for scientific research in order to outline the pathogenesis of different diseases. However, in future it could be advisable to include such studies with validated value in the routine investigation as technically the amount of tissue in liver biopsy is sufficient.

Cytokine expression can be analysed, e.g., TGF, EGFR and others [25]. When studying inter‐ leukin-6 (IL-6) expression in liver biopsies, higher IL-6 expression was found in non-alcohol‐ ic steatohepatitis than in steatosis. Correlation between IL-6 expression and degree of inflammation and stage of fibrosis was detected as well [53]. Due to the complex nature of cytokine action, wide spectrum of different molecules and their receptors must be analysed in details in order to avoid insignificant or contradictory results. This leads to a clear-cut ne‐ cessity for virtual microscopy and digital image analysis. Toll-like receptor-4 (TLR4) expres‐ sion can be analysed in liver biopsy by immunohistochemistry. The expression of TLR4 has been shown in hepatic progenitor cells and interlobular bile duct epithelium in correlation with stage of liver disease, grade of liver inflammation and activity of portal/septal myofi‐ broblasts [54]. The expression of interferon stimulated gene 15 can be analysed by IHC at protein level; up-regulation in hepatocytes is more pronounced in patients not responding to interferon / ribavirin treatment in contrast to predominant expression in Kuppfer cells in treatment responders [55]. Proteomic studies including immunohistochemistry in liver biop‐ sy have targeted cell structure-associated proteins - actin, tropomyosin, transgelin and hu‐ man microfibril-associated protein 4 in order to identify biomarkers of liver cirrhosis [56]. COX-2 is over-expressed in chronic hepatitis C and the expression decreases following treat‐ ment with interferon alpha regardless of sustained virological response [57]. Increased en‐ doglin and TGF beta 1 expression is significantly associated with progressive hepatic fibrosis in chronic viral hepatitis C [58].

tolerated level of 32% [40] or approximately 36% in liver cirrhosis necessitating liver transplantation [24]. Thus, three approaches are combined: the outlines of regular struc‐ tures as vacuoles are characterised by traditional, non-fractal geometry, the area of fibro‐ sis and parenchyma are detected using the traditional measurements corrected by the fractal dimension, and the tectonic index is based on the relationships between the Eucli‐ dean and fractal dimensions of liver tissue. One of the many positive features of this sys‐ tem is the ability to generate continuous scalar variables. When analysing dynamics in repeated liver biopsies by scalar data, naturally, less biopsies are characterised as lacking

Although fractal concept is used in medicine, including at least microscopy, neuroscience and ophthalmology as well as automated measurements not limited to pathology [49, 51, 52], the study described in reference [40] is remarkable as it is highly sophisticated and prac‐ tical; it is understandable that the research group considers their machine as an intelligent

The diagnostic evaluation of liver biopsy is mostly based on panel of histochemical stains including hematoxylin-eosin [2], Masson's trichrome [3], PAS [5] and Perl's [1] stains as well as others by necessity. These visualisation techniques should be comple‐ mented by various "- omics" tools [27] to gain more data on the function of liver cells. The cytokines, inflammatory mediators, viral proteins, cell cycle proteins and apoptosis markers can be detected; metabolic pathways can be investigated as well. At present, most or proteomic and genetic studies are carried out for scientific research in order to outline the pathogenesis of different diseases. However, in future it could be advisable to include such studies with validated value in the routine investigation as technically the

Cytokine expression can be analysed, e.g., TGF, EGFR and others [25]. When studying inter‐ leukin-6 (IL-6) expression in liver biopsies, higher IL-6 expression was found in non-alcohol‐ ic steatohepatitis than in steatosis. Correlation between IL-6 expression and degree of inflammation and stage of fibrosis was detected as well [53]. Due to the complex nature of cytokine action, wide spectrum of different molecules and their receptors must be analysed in details in order to avoid insignificant or contradictory results. This leads to a clear-cut ne‐ cessity for virtual microscopy and digital image analysis. Toll-like receptor-4 (TLR4) expres‐ sion can be analysed in liver biopsy by immunohistochemistry. The expression of TLR4 has been shown in hepatic progenitor cells and interlobular bile duct epithelium in correlation with stage of liver disease, grade of liver inflammation and activity of portal/septal myofi‐ broblasts [54]. The expression of interferon stimulated gene 15 can be analysed by IHC at protein level; up-regulation in hepatocytes is more pronounced in patients not responding to interferon / ribavirin treatment in contrast to predominant expression in Kuppfer cells in treatment responders [55]. Proteomic studies including immunohistochemistry in liver biop‐

collaborator – and this is exactly the way how future biopsy analysis should proceed.

**7. Functional liver tissue analysis in biopsy**

amount of tissue in liver biopsy is sufficient.

significant changes.

270 Liver Biopsy – Indications, Procedures, Results

Cell cycle analysis can add valuable information [59]; digital image analysis should be add‐ ed in the logistics again. Arrested cell cycle status has been demonstrated in chronic hepati‐ tis C infection analysing the expression of mini-chromosome maintenance protein-2 as higher sensitivity proliferation marker, G1 phase marker cyclin D1, S phase marker cyclin A, cell cycle regulators p21 and p53, apoptotic protein caspase 3 and anti-apoptotic protein Bcl-2 [60, 61]. When analysing liver biopsies from patients with chronic viral hepatitis C, higher G1 and lower S phase fractions has been found also by Werling *et al*., employing im‐ age analysis method [59]. Apoptosis-related pathways can be explorated including evalua‐ tion of Bax, Bcl-xL and Bcl-2 proteins [62]. Thus, hepatitis C virus infection can deregulate the cellular processes [63] and it can be practical to reveal the way and degree of the regula‐ tory shift.

Viral antigens including hepatitis C antigen can be detected in liver tissue by immunohisto‐ chemistry [64]; the finding can be helpful in cases with difficult differential diagnosis or combined liver pathology. The association of expression pattern with fibrosis may suggest pathogenetically important information as well [64].

Metabolic pathways can be evaluated in liver biopsy. For instance, widespread expression of vitamin D receptor has been shown in the hepatocytes and inflammatory cells in case of chronic liver disease including non-alcoholic steatohepatitis and chronic viral hepatitis C. The expression decreases as the liver histology is damaged [65].

Inflammatory cells are as important components in diffuse liver disease as the hepatocytes. Thus, higher numbers of intrahepatic follicular T-helper lymphocytes in conjunction with IL28B polymorphism analysis is found to be strongly predictive of treatment response using pegylated interferon and ribavirin [66]. CD4+ regulatory T cells can be evaluated [67].

Logistic structures have been implemented to develop next generation toolkits for automat‐ ed image analysis to enable quantification of molecular markers. The group of researchers [27] have collaborated within open source image analysis project [68] to reach effective out‐ put by combination of quantitative analysis, multiplex quantum dot (nanoparticle) staining and high resolution whole slide imaging to detect nine different fluorescent signals for mul‐ tiple antigens [27].

DNA microarray technology has enabled genome-wide analysis of gene transcript levels. This technology has been applied in order to compare gene expression profiles at different stages of chronic hepatitis C and hepatocellular carcinoma in the setting of hepatitis virus C infection [63, 69]. Hundreds of genes involved in carcinogenesis, cell growth, proliferation and death are differently expressed in advanced viral hepatitis C in comparison to early vi‐ ral hepatitis C or non-viral hepatitis [63]. In chronic hepatitis C, the up-regulation involves genes related to metabolism and immune responses. In hepatocellular carcinoma arising in hepatitis C patients, genes associated with cell cycle, growth, proliferation and apoptosis are up-regulated [69]. Chronic hepatitis B and autoimmune liver disease have been studied by this technology as well [70]. In advanced chronic viral hepatitis B, genes associated with ex‐ tracellular matrix turnover, cell growth and DNA repair are up-regulated but the expression of genes regulating complement activation and innate immune response is decreased. In early disease stages, the gene expression is different in case of chronic viral hepatitis B, auto‐ immune hepatitis and primary biliary cirrhosis. Chronic viral hepatitis B is associated with expression of genes considering chemotaxis and cell homeostasis; autoimmune hepatitis – with down-regulation of genes associated with protein binding, but primary biliary cirrhosis in early stages involves the actin and myosin gene expression. As chronic viral hepatitis B progresses, the expression of genes regarding signalling pathway, cell communication, col‐ lagen turnover, chemokine ligands and metallothionein changes [70]. The findings are of major interest displaying the pathogenesis of different inflammatory liver diseases and neo‐ plastic transformation. Diagnostic consequences should follow soon as the differential diag‐ nosis of inflammatory liver diseases regarding aetiology can represent a difficult task.

tential hepatotoxic effect, with identification of viruses, as well as with determination of in‐

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, Valda Zalcmane1

Future Aspects of Liver Biopsy: From Reality to Mathematical Basis of Virtual Microscopy

, Andrejs Ivanovs1

http://dx.doi.org/10.5772/52753

and

273

, Boriss Strumfs3

fluence of various environmental hazards.

**Author details**

Valentina Sondore2

**References**

p243-267.

inburgh, 2002. p125-138.

2002. p139-162.

1:431-435.

tol, 1991; 13:372-374.

Ludmila Viksna1,2, Ilze Strumfa1

1 Riga Stradins University, Riga, Latvia

2 Riga Eastern Clinical University Hospital, Riga, Latvia

3 Latvian Institute of Organic Synthesis, Riga, Latvia

The level of mRNA can be post-transciptionally regulated by micro RNA (miRNA). The reg‐ ulation of biological processes by miRNA is shown also in case of such canonical diffuse liv‐ er disease as chronic viral hepatitis C. Technological studies have been conducted using biopsy material [71]. Transcriptome analysis has shown prognostic value, e.g., in order to predict the severity of fibrosis progression after liver transplantation in recurrent viral hepa‐ titis C patients [72].
