**2. Morphological evaluation of liver: Today's reality**

Despite the fact that the histological assessment of liver tissues plays an essential role in the diagnosis of liver diseases and the histological conclusion serves quite often as a basis for

© 2012 Viksna et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

establishing the diagnosis, there are factors or reasons to be taken into consideration which can negatively influence the results obtained in morphological evaluation of liver biopsy.

Further we will have a look at each factor separately. The first reason which may significant‐ ly affect the final result is the incidental character of biopsy specimen collection by means of "blind" biopsy. In case of diffuse liver damage, it is important to obtain liver specimen from a representative site (which is not subcapsular) or under ultrasonographic (USG) control. If the liver specimen is obtained during invasive procedure (laparoscopy or open abdominal surgery), it is of high importance to give information about preferable biopsy site to the col‐ league obtaining liver specimen. The liver specimens obtained during surgery are certainly more targeted. More or less qualitative methodological performance of tissue collection may

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Presuming that the biopsy specimen is obtained from the site typical for the certain liver pathology, one more important issue is the quality of biopsy specimens' fixation and slicing.

The aspect of "special" tissue staining must also be looked at, because in case of absence of examination request or list of preliminary diagnosis provided by clinician, that emphasizes the need for particular staining, morphologist is unable to give an adequate diagnostic as‐ sessment of biopsy specimen. Thus diagnosis like haemochromatosis and other pathologies

The next factor, i.e., selection of certain section out of the whole biopsy specimen, is an issue arising only in case if the biopsy specimen is not examined throughout or along its horizon‐ tal length. The cross-sectioning gives the chance to analyze tissues on different "depths" or

The technical condition or quality of the microscope and number of viewable visual fields are to be considered seriously. Nowadays, the usual practice of the pathologist is a general over‐ view of the material to gain insight into overall picture, noticing the most typical and impor‐ tant peculiarities. Inaccuracies can occur if only some separate visual fields are examined.

The subjective component of the morphological assessment of liver specimens and interpre‐ tation of the observed changes and their compliance or adherence to one or the other pathol‐ ogy is essential also. The problem could be the qualification and experience of clinician to put together or combine visual insight in the particular biopsy specimen and clinical diagno‐ sis made up of biochemical, immunological and genetic parameters, and to use the interpre‐

Selection of morphological or histological evaluation scale is significant. These scales are very advantageous for standardizing expert's assessment, converting it into measurable characteristics and helping the clinician to make final decision about patient's diagnosis. In case of light microscopy the issue of selection of evaluation scale is a factor with up to 100% error probability. For example, the use of the Knodell scale for patient with steatohepatosis, HAI = 0, leads to incorrect conclusion that the patient is healthy, especially if the biochemi‐

If in addition the electron microscopic investigation of sequential liver biopsy specimens are done, obtained results and conclusions are also affected by the whole process of the above men‐ tioned biopsy specimen collection and processing. The electron microscopy is currently consid‐

tation of morphologist properly for establishing the diagnosis.

also cause certain imperfections affecting quality of specimen evaluation.

known as "storage diseases" can be missed.

"levels" of the biopsy specimen.

cal parameters of blood are not altered.

We have "assessed" the percentage of each factor's influence on the final result– description and conclusion regarding diagnosis, where "0" is considered a factor that does not affect the evaluation and its outcome, but "100%" – the factor which actually hinders the correct diag‐ nosis of the disease. The factors that may affect the liver tissue morphological assessment and diagnosis of disease are summarized in Table 1.


\* 100% - affecting

0% - not affecting

**Table 1.** Factors affecting liver tissue morphologic assessment

Further we will have a look at each factor separately. The first reason which may significant‐ ly affect the final result is the incidental character of biopsy specimen collection by means of "blind" biopsy. In case of diffuse liver damage, it is important to obtain liver specimen from a representative site (which is not subcapsular) or under ultrasonographic (USG) control. If the liver specimen is obtained during invasive procedure (laparoscopy or open abdominal surgery), it is of high importance to give information about preferable biopsy site to the col‐ league obtaining liver specimen. The liver specimens obtained during surgery are certainly more targeted. More or less qualitative methodological performance of tissue collection may also cause certain imperfections affecting quality of specimen evaluation.

establishing the diagnosis, there are factors or reasons to be taken into consideration which can negatively influence the results obtained in morphological evaluation of liver biopsy.

We have "assessed" the percentage of each factor's influence on the final result– description and conclusion regarding diagnosis, where "0" is considered a factor that does not affect the evaluation and its outcome, but "100%" – the factor which actually hinders the correct diag‐ nosis of the disease. The factors that may affect the liver tissue morphological assessment

1. Biopsy site selection in liver tissues 100 – 0 It can be completely non-

**Evaluation of impact on final outcome in % \***

100 – 0

30 – 0

6. Number of viewable visual fields 80 – 0 Inaccuracies can occur if only some

**Comments**

representative site, such as subcapsular

Chemical environment (composition) and temperature of fixation can affect

error theoretically is not possible

If the sample is not stained for the reason to label a certain substance, e.g., Fe, the error can reach 100%

Thick or disrupted tissue sections can hinder the pathology from the observer. The thickness should not

separate visual fields are examined

Incomplete quality of optical system can hinder the pathology from the

If the specimen where the basic pathology relates to fatty changes (steatosis) is assessed according to Knodell scale, then the assessment is inadequate if compared to the actual

exceed 3-4 micrometres.

observer.

liver tissue damage

the biopsy specimen

50 – 0 If the whole sample is used, then the

and diagnosis of disease are summarized in Table 1.

**morphologic assessment**

Selection of certain section out of the

4. Selection and quality of staining (panel of visualization methods) of the sample

5. Quality of biopsy specimen sections or

2. Fixation and transfer of biopsy sample 100 – 0

7. Technical condition of the microscope 80 – 0

8. Selection of evaluation scale 100 – 0

**Table 1.** Factors affecting liver tissue morphologic assessment

**No. Factors affecting liver tissue**

258 Liver Biopsy – Indications, Procedures, Results

3.

whole sample

microtomy

\* 100% - affecting 0% - not affecting

Presuming that the biopsy specimen is obtained from the site typical for the certain liver pathology, one more important issue is the quality of biopsy specimens' fixation and slicing.

The aspect of "special" tissue staining must also be looked at, because in case of absence of examination request or list of preliminary diagnosis provided by clinician, that emphasizes the need for particular staining, morphologist is unable to give an adequate diagnostic as‐ sessment of biopsy specimen. Thus diagnosis like haemochromatosis and other pathologies known as "storage diseases" can be missed.

The next factor, i.e., selection of certain section out of the whole biopsy specimen, is an issue arising only in case if the biopsy specimen is not examined throughout or along its horizon‐ tal length. The cross-sectioning gives the chance to analyze tissues on different "depths" or "levels" of the biopsy specimen.

The technical condition or quality of the microscope and number of viewable visual fields are to be considered seriously. Nowadays, the usual practice of the pathologist is a general over‐ view of the material to gain insight into overall picture, noticing the most typical and impor‐ tant peculiarities. Inaccuracies can occur if only some separate visual fields are examined.

The subjective component of the morphological assessment of liver specimens and interpre‐ tation of the observed changes and their compliance or adherence to one or the other pathol‐ ogy is essential also. The problem could be the qualification and experience of clinician to put together or combine visual insight in the particular biopsy specimen and clinical diagno‐ sis made up of biochemical, immunological and genetic parameters, and to use the interpre‐ tation of morphologist properly for establishing the diagnosis.

Selection of morphological or histological evaluation scale is significant. These scales are very advantageous for standardizing expert's assessment, converting it into measurable characteristics and helping the clinician to make final decision about patient's diagnosis. In case of light microscopy the issue of selection of evaluation scale is a factor with up to 100% error probability. For example, the use of the Knodell scale for patient with steatohepatosis, HAI = 0, leads to incorrect conclusion that the patient is healthy, especially if the biochemi‐ cal parameters of blood are not altered.

If in addition the electron microscopic investigation of sequential liver biopsy specimens are done, obtained results and conclusions are also affected by the whole process of the above men‐ tioned biopsy specimen collection and processing. The electron microscopy is currently consid‐ ered as an auxiliary method or technique, yet in the age of high-tech medicine, processes ongoing on the level of organelles are the ones which by characteristic ultrastructural changes frequently refer to or indicate a particular pathology. The following must be strictly observed in electron microscopy: 1) liver tissue sampling and slicing into 1 mm3 pieces without mechanical‐ ly squeezing them and immediate immersion in fixing solution; 2) chemical composition of fix‐ ing solution, temperature, sample fixation and rinsing time; 3) embedding of liver tissue samples in mixture of epoxy resins in accordance with polymerization time of these resins; 4) quality of sample cutting with ultramicrotome and contrasting with uranyl acetate and lead cit‐ rate; 5) all cells and their organelles visible in the ultra-thin slices under the electron microscope have to be examined. It should be noted that resolution of transmission electron microscope (TEM) is within the range of 0.2 to 2 nm and resolution of scanning electron microscope is 4 nm.

The virtual microscopy can be performed in two different ways. Interactive virtual micro‐ scopy by whole slide imaging leaves the conclusion in the hands of pathologist. It changes significantly the working tools from optical microscopy and subjective decisions to comput‐ er screen and objective measurements. The automated virtual microscopy is even more ex‐

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In liver pathology, the software develops regarding assessment of steatosis [17-19] and fib‐

Regarding liver ultrastructure, morphometric evaluation of hepatocyte volume can have prognostic significance predicting survival as shown in liver cirrhosis associated with portal hypertension [25]. Morphometric analysis of liver parenchyma in different alcohol-related pathologic conditions has been tested with good results [26]. Thus, changes in the volume fraction of parenchymal interstitial space and in the surface density of hepatocyte plasma membrane, rough endoplasmic reticulum and outer mitochondrial membrane can be of im‐ portance for distinguishing between cirrhosis and non-cirrhotic states. Hepatocyte nuclear volume fraction measurement can predict the survival in case of cirrhosis. Interestingly, few images are necessary to perform these measurements thus helping to characterise even

Combination of multiplex quantum dot immunostaining with high resolution whole-slide

At present, the two most frequently studied targets for computer-assisted and/or digital im‐

Among Western population, liver steatosis is a frequent finding [28-29] as it is associated with such common factors as chronic viral hepatitis [19], alcohol drinking, diabetes mellitus or obesity [17]. It has been considered a risk factor for liver fibrosis [18, 19]. Steatosis, includ‐ ing non-alcoholic steatohepatitis [19] has become an important target in diagnostics and sci‐ entific research therefore highly reproducible measurements are necessary to evaluate the course of disease, outcome and effect of treatment. The biopsy is still considered a gold standard in the diagnosis and assessment of steatosis as the imaging including ultrasonogra‐ phy, computed tomography and magnetic resonance imaging can be affected by lower sen‐ sitivity [17, 30]. The severity of steatosis in liver biopsies can be graded by several semiquantitative systems (Table 2) assessing the eyeballed proportion of affected cells [30-35].

The present semiquantitative estimates are subjective and limit the possibilities of statistic analysis [18]. Numerical value, expressing the exact percentage of affected cells would be more reliable if an adequate biopsy is analysed. Such measurement is possible, especially in computer-assisted way, but it would require architecturally arranged count of nuclei and fat vacuoles per biopsy. Thus, the measurement would be time-consuming and accordingly ex‐ pensive. On the other hand, steatosis is relatively easy target for digital quantification of the

citing as computer system should evaluate the diagnoses [14].

scarce tissue material [26].

rosis [20-24]. Necroinflammatory changes can be quantified as well [16].

digital imaging and automated image analysis has been described [27].

age analysis in liver biopsies include steatosis and fibrosis.

**4. Digital assessment of liver steatosis**
