# Quantification of Liver Fibrosis--A Comparative Study
## 1 Introduction  
Early diagnosis and accurate staging have always been considered essential for the determination of a treatment strategy. Recently, nearly 35 million people globally have died due to hepatic diseases [1]. During the last few decades, the clinical management of chronic liver disease (CLD) has been increasingly focused on the prevention of the development or progression of fibrosis. Liver fibrosis develops as a result of chronic damage, which eventually leads to cirrhosis, marking the final stage of liver disease. Technically, thick collagen fibers have been targeted as the main source of fibrosis (Figure 1). During chronic hepatic injury, stellate hepatocytes are activated and transformed into a myofibroblast-like phenotype forming an extracellular matrix (ECM). The main causes of stimulating these cells include tissue inflammation, cytokine production from injured parenchymal cells, and disruption of the extracellular matrix [2].  
Fibrosis estimation is a standard procedure, where liver biopsies are formalin-fixed and paraffin-embedded. Thereafter, the specimens are commonly cut to a thickness of 4 \(\upmu\)m and stained using several dyes. The most common dyes used for fibrosis estimation are the Masson's trichrome (MT) and Sirius red (SR). The above dyes bind selectively to liver collagen, allowing for fibrosis quantitation. Liver biopsy has become the gold standard in most clinical diagnostic efforts [3]. The needle biopsy specimens are examined from pathologists in order to determine the stage of the liver disease. The specimens are staged and graded according to a scoring system, with respect to the zonation as well as the number and the width of septa. The score represents categories of increasing severity based on a combination of the fibrosis amount and fibrous forms such as septa, bridges and  
The Knodell HAI has four stages (0 to 3), the Scheuer and METAVIR use five stages (0 to 4), and the Ishak HAI (Figure 2) refers to seven stages (0 to 6). These scoring systems have been as well introduced as standards for all semi-quantitative methods in the classification of the chronic active hepatitis (CAH) [9]. The last stage of the [PERSON], Scheuer, and METAVIR scoring systems describes cirrhosis, while in the Ishak HAI, stage 5 indicates incomplete cirrhosis with occasionally detected nodules and stage 6 definite cirrhosis, respectively. The main disadvantage of the diagnosis based on semi-quantitative systems is the subjectivity of the observer, commonly referred to as \"intra-observer\" variability.  
A quantitative approach for fibrosis scoring is the measurement of the collagen proportional area (CPA), which is the ratio of the collagen area to the hepatic tissue area in microscopy liver biopsy images using digital image analysis (DIA) techniques. Several studies have been presented in the literature in the past three and a half decades, with the majority focusing on the clinical validation of CPA by investigating the correlation of semi-quantitative scoring systems (SSS) with the CPA obtained from DIA.  
This study aims to identify the strengths and limitations of DIA techniques and to point out \"gray-areas\" as diagnostic obstacles in the major findings classification landscape, which tend to make unreliable the interpretation of medical results. The rest of the paper is structured as follows: Section 2 focuses on the search steps followed to collect all reviewed research materials, with image analysis methods aiming at identifying fibrotic areas of collagen accumulation in liver biopsy specimens (Figure 3). Subsequently, Section 3 describes the used methodological approach (divided into subsections). The results produced from each of the studies presented in the literature are discussed in Section 4, with the concluding remarks listed in Section 5.  
Figure 2: Illustration of the Ishak system stages (0–6) on histologically stained Sirius red (SR) tissue sections.