**6. Digital biopsy analysis for inflammatory liver lesion: Future begins today**

The incorporation of Mandelbrot's fractal geometry [48] into the digital evaluation of liver biopsy for chronic hepatitis has brought revolutionary changes [40].

The short description of fractal is provided in Table 3; detailed characteristics can be found in recent reviews [49].


**Table 3.** The characteristics of fractals

When analysing liver steatosis, the observations of higher accuracy in resin-embedded sam‐ ples [18] request more technological progress in order to create methodology for easy use in

Digital stereological point counting has been employed in liver steatosis evaluation as well [33]. The researchers have observed the same fact that manual semiquantitative assessment tends to be significantly higher. The lack of precision in manual evaluation can be related to

Some researchers have also come to the conclusion that automated assessment of liver stea‐ tosis is more time-consuming than manual [30]. The time input for digital measurement is found to be threefold greater than for manual evaluation [19]. Although this opinion is based on trustable experience, half of the problem is solved already as the whole slide imag‐ ing eliminates the need to choose appropriate number of representative fields submitted for analysis and the necessity for human participation in the obtaining and archiving of digital images. Besides the whole slide imaging, the degree of automatisation must be further in‐ creased: optimal software abolishes the manual correction of object inclusion into measure‐ ments. However, this deserves morphologically correct mathematical model. Other groups

The computer-aided assessment of necroinflammatory processes in chronic viral hepatitis has been tested. To perform this, immunohistochemical visualisation is necessary in order to high‐ light inflammatory cells. The application of immunohistochemistry increases the expenses. This drawback can be counterbalanced by gains of rapid measurement, resulting in rigorous results expressed in scalar numbers as well as by complete characteristics more exactly reflect‐

The assessment of hepatic fibrosis and the closely related architectural deformities as bridging fibrosis and liver cirrhosis have important role in the diagnostics, treatment and prognostic evaluation of chronic liver diseases [24]. The studies of liver fibrosis are facilitated by standard use of special stains for the routine evaluation of liver biopsies in case of diffuse liver disease. Masson's trichrome is an efficient method to highlight fibrosis [3]. The sharp contrast between blue collagen and red parenchyma allows visualisation of even small excess amounts of colla‐ gen [23]. Sirius red stain has also been employed [21, 40]; it has the benefit of selective staining of collagen but not proteoglycans [22]. Not surprisingly, comparatively many authors have ap‐ plied digital image analysis to quantify fibrosis in liver tissue [24]. Validation studies of com‐ puter-assisted morphometry have also been performed [21]. Besides the well-developed methodology including software, the application of computer analysis has resulted in exact numerical data allowing detection of interesting biological relationships. For instance, the cor‐ relation of fibrosis burden with end-stage liver disease score, serum total bilirubin and interna‐ tional standard ratio of prothrombin has been shown in hepatitis B-related decompensated cirrhosis. Thus, the correlation between the amount of connective tissue in cirrhotic liver and

the physiology of vision and processing of the visual information [19, 39].

have considered computer-aided morphometry to be fast and objective [16].

**5. Digital assessment of inflammation in liver biopsy**

ing the status of the whole organ [16].

routine samples.

264 Liver Biopsy – Indications, Procedures, Results

Hurst's exponent is another albeit related mathematical construct with major meaning in the digital analysis of liver biopsy. It was first used to study the variation in water flow in Nile ba‐ sin during the construction of the Aswan dam [16, 50]. In general, it can be used to detect the ir‐ regularity – a key parameter analysing the activity of inflammation in the liver as the active inflammation manifests with periportal piecemeal necrosis causing irregularity in the normal‐ ly smooth border of portal field. Hurst's exponent also can be detected by fractal mathematics. It can describe quantitatively the deviation from smooth contour in natural fractal objects.

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To detect the border of inflammatory cell cluster, Delaunay's triangulation can be used with success. In general, Delaunay's triangulation involves set of points in such way that no point is inside the circle drawn through 3 points. It maximizes the minimum angle avoiding narrow triangles. If circle drawn through 2 input points contain the third point in the outside, these points form Delaunay's triangle. The method can be used to mesh the space. By this triangula‐ tion, lines were drawn in the scanned image of liver biopsy through each pair of adjacent in‐ flammatory cells resulting in network of triangles showing common border. The most external triangle short sides formed the border of inflammatory cell infiltrate. The triangle side was de‐ fined as appropriately short if it was equal of less than 20 microns based on empiric analysis. After the cluster has been outlined, both the amount (by area) of inflammatory cells and the

The mathematical basis of so-called geometry of irregularity (Figure 2-3) has allowed to detect the amount of residual liver parenchyma, inflammation (Figure 4-5) and fibrosis (Figure 6-7) as well as to provide index characterising the appropriateness of liver tissue structure (named tec‐

**Figure 4.** Irregular outline (arrowheads) of portal field in chronic active hepatitis. Haematoxylin-eosin stain, original

border irregularity and area of cluster-affected tissue can be evaluated [16].

tonic index by the authors).

magnification 100x

**Figure 2.** Highly irregular structure of biological object. Use of Mandelbrot's fractal geometry is suggested to describe targets with remarkable degree of complexity and irregularity. Note also the similarity of complex, branching outline with Figures 4 and 5

**Figure 3.** Retained irregularity of the biological structure at higher magnification: note the remarkable similarity with Figure 2. The persisting complexity at different levels of magnification is another feature suggesting the necessity for fractal analysis. The inflammation in liver biopsy (shown in Figures 4 and 5) depicts analogous features

Hurst's exponent is another albeit related mathematical construct with major meaning in the digital analysis of liver biopsy. It was first used to study the variation in water flow in Nile ba‐ sin during the construction of the Aswan dam [16, 50]. In general, it can be used to detect the ir‐ regularity – a key parameter analysing the activity of inflammation in the liver as the active inflammation manifests with periportal piecemeal necrosis causing irregularity in the normal‐ ly smooth border of portal field. Hurst's exponent also can be detected by fractal mathematics. It can describe quantitatively the deviation from smooth contour in natural fractal objects.

To detect the border of inflammatory cell cluster, Delaunay's triangulation can be used with success. In general, Delaunay's triangulation involves set of points in such way that no point is inside the circle drawn through 3 points. It maximizes the minimum angle avoiding narrow triangles. If circle drawn through 2 input points contain the third point in the outside, these points form Delaunay's triangle. The method can be used to mesh the space. By this triangula‐ tion, lines were drawn in the scanned image of liver biopsy through each pair of adjacent in‐ flammatory cells resulting in network of triangles showing common border. The most external triangle short sides formed the border of inflammatory cell infiltrate. The triangle side was de‐ fined as appropriately short if it was equal of less than 20 microns based on empiric analysis. After the cluster has been outlined, both the amount (by area) of inflammatory cells and the border irregularity and area of cluster-affected tissue can be evaluated [16].

The mathematical basis of so-called geometry of irregularity (Figure 2-3) has allowed to detect the amount of residual liver parenchyma, inflammation (Figure 4-5) and fibrosis (Figure 6-7) as well as to provide index characterising the appropriateness of liver tissue structure (named tec‐ tonic index by the authors).

**Figure 2.** Highly irregular structure of biological object. Use of Mandelbrot's fractal geometry is suggested to describe targets with remarkable degree of complexity and irregularity. Note also the similarity of complex, branching outline

**Figure 3.** Retained irregularity of the biological structure at higher magnification: note the remarkable similarity with Figure 2. The persisting complexity at different levels of magnification is another feature suggesting the necessity for

fractal analysis. The inflammation in liver biopsy (shown in Figures 4 and 5) depicts analogous features

with Figures 4 and 5

266 Liver Biopsy – Indications, Procedures, Results

**Figure 4.** Irregular outline (arrowheads) of portal field in chronic active hepatitis. Haematoxylin-eosin stain, original magnification 100x

**Figure 5.** Branching pattern (arrowheads) of periportal inflammatory infiltrate. Note the remarkable similarity with Figure 4 analogous to the relationship between Figures 2-3. The fractal nature of inflammation is thus highlighted. Haematoxylin-eosin stain, original magnification 400x

**Figure 7.** Branching pattern of connective tissue fields in arachnoid liver fibrosis (arrowhead). Masson's trichrome

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The Dioguardi Histological Metriser machine, described in reference [40] is able to pro‐ duce measurements and even simple diagnoses, working with reasonable speed. The rel‐ evant equipment ensures microscope focusing and full slide scanning, and determines the above mentioned parameters excluding any unfilled spaces as vessels, sinusoids, bili‐ ary ducts and artifactual holes. The system is able to identify and exclude the Glisson's capsule from the analysis. Colour thresholds are used to select the areas of interest. The inflammatory cells are identified by immunohistochemical visualisation of leukocyte common antigen. For the analysis, the inflammatory cell clusters are outlined by imagi‐ nary line connecting the centres of the outermost cells; after that the area of clusters is measured. Thinking in the usual terms, the portal and periportal infiltrates are character‐ ised by this measurement; the portal fibrosis also can influence this measurement provid‐ ing homing space for inflammatory infiltrate. The area of extra-cluster inflammatory cells is measured separately; these could mostly correspond to intralobular infiltrate. When analysing fibrosis, area of fibrotic tissue is measured. The wrinkledness is detected as the ratio between the perimeter and area of an object. As portal field in healthy liver is smooth, the concept of wrinkledness is an efficient way to detect periportal inflammation and portal fibrosis. The irregularity of collagen islets necessitates the correction by fractal dimension; the fibrotic foci are considered truncated planar fractals. The residual paren‐ chyma is characterised by the tissue area that is not occupied by inflammatory cells and fibrosis. Finally, the loss of order is characterised mathematically. In order to characterise the course of the disease in analogue with the usual staging, the individual fibrosis sca‐ lar is compared with the curve of fibrosis development over the course of disease detect‐ ing the percentage of the disease course before collagen deposition reaches the maximal

stain, original magnification 400x

**Figure 6.** Branching outline of connective tissue fields in liver cirrhosis. Note both the large areas of connective tissue (star) and the thin septa (arrowheads). Masson's trichrome stain, original magnification 100x

**Figure 7.** Branching pattern of connective tissue fields in arachnoid liver fibrosis (arrowhead). Masson's trichrome stain, original magnification 400x

**Figure 5.** Branching pattern (arrowheads) of periportal inflammatory infiltrate. Note the remarkable similarity with Figure 4 analogous to the relationship between Figures 2-3. The fractal nature of inflammation is thus highlighted.

**Figure 6.** Branching outline of connective tissue fields in liver cirrhosis. Note both the large areas of connective tissue

(star) and the thin septa (arrowheads). Masson's trichrome stain, original magnification 100x

Haematoxylin-eosin stain, original magnification 400x

268 Liver Biopsy – Indications, Procedures, Results

The Dioguardi Histological Metriser machine, described in reference [40] is able to pro‐ duce measurements and even simple diagnoses, working with reasonable speed. The rel‐ evant equipment ensures microscope focusing and full slide scanning, and determines the above mentioned parameters excluding any unfilled spaces as vessels, sinusoids, bili‐ ary ducts and artifactual holes. The system is able to identify and exclude the Glisson's capsule from the analysis. Colour thresholds are used to select the areas of interest. The inflammatory cells are identified by immunohistochemical visualisation of leukocyte common antigen. For the analysis, the inflammatory cell clusters are outlined by imagi‐ nary line connecting the centres of the outermost cells; after that the area of clusters is measured. Thinking in the usual terms, the portal and periportal infiltrates are character‐ ised by this measurement; the portal fibrosis also can influence this measurement provid‐ ing homing space for inflammatory infiltrate. The area of extra-cluster inflammatory cells is measured separately; these could mostly correspond to intralobular infiltrate. When analysing fibrosis, area of fibrotic tissue is measured. The wrinkledness is detected as the ratio between the perimeter and area of an object. As portal field in healthy liver is smooth, the concept of wrinkledness is an efficient way to detect periportal inflammation and portal fibrosis. The irregularity of collagen islets necessitates the correction by fractal dimension; the fibrotic foci are considered truncated planar fractals. The residual paren‐ chyma is characterised by the tissue area that is not occupied by inflammatory cells and fibrosis. Finally, the loss of order is characterised mathematically. In order to characterise the course of the disease in analogue with the usual staging, the individual fibrosis sca‐ lar is compared with the curve of fibrosis development over the course of disease detect‐ ing the percentage of the disease course before collagen deposition reaches the maximal 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 significant changes.

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

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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‐

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

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.

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‐

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

fibrosis in chronic viral hepatitis C [58].

pathogenetically important information as well [64].

The expression decreases as the liver histology is damaged [65].

tory shift.

tiple antigens [27].

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