**8. Non-invasive tests for liver disease and assessment of fibrosis and cirrhosis**

Limitations of biopsy have led clinical investigators to study alternative methods to in‐ vestigate liver disease especially for the assessment of liver fibrosis. Since fibrosis is of sufficient importance in chronic liver disease and because it progresses to cirrhosis it is frequently used as the outcome and prognostic variable in clinical studies. Hepatic fib‐ rosis is currently viewed as a dynamic process that may regress after successful treat‐ ment of chronic liver diseases. Serum markers, such as non-invasive markers, offer an attractive alternative. They are objective, allowing a dynamic calibration of fibrosis, can be performed repeatedly, are more cost effective and many of them are performed as a routine analysis [109].

**Indirect markers of liver fibrosis:** among them, the AST to Platelet Ratio Index (APRI), combines aspartate amiotransferase (AST) with platelet count. It was used in several studies conducted in cohorts of patients with hepatitis C and showed a rather good di‐ agnostic performance and reproducibility, [110] particularly for cirrhosis. Forns and col‐ leagues reported a fibrosis index (Forns' index) based on platelet count, γ-glutamyl transferase (GGT), and cholesterol levels [111]. It is rather good for excluding significant fibrosis, but only average for diagnosing significant fibrosis. However, one important limit of both APRI and Forns' indexes are that they leave almost half of the patients unclassified.

Another widely investigated combination set of noninvasive markers of liver fibrosis is the Fibrotest; a combination of five blood tests based on a mathematical formula: GGT, biliru‐ bin, haptoglobin, apolipoprotein A1, alfa2 macroglobulin adjusted for gender and age. Ac‐ cording to the investigators, it could exclude cases with significant fibrosis (METAVIR > F2), having 100% of negative predictive value, and more than 90% positive predictive value, us‐ ing liver biopsy as a reference [112].

#### **8.1. Elastography (FibroScan)**

tract, and liver. It is characterized by degenerative bile duct lesions with some degree of mononuclear inflammation. Cholestasis may be present. Chronic GVHD is a multiorgan process that develops 80 to 400 days after transplantation and is often preceded by acute GVHD. The changes in the liver are similar to those in acute disease, but the ducts show more prominent changes and are likely to be reduced in number or destroyed. A prominent

Liver biopsies detect silent donor disease in potential living liver donors, especially patients suffering subclinical non-alcoholic fatty liver disease (NAFLD). The contribution of liver bi‐ opsy or even the need to perform this, when a potential donor is being evaluated is a contro‐ versial issue [107]. In the University of Pittsburgh Medical Center a retrospective study of the histopathologic examination and diagnoses of 284 patients, who were evaluated as living do‐ nors from 2001 to 2005 was carried out. Hepatic histology was correlated with liver injury tests and with demographic characteristics in an otherwise normal healthy population. A mi‐ nority (n=119; 42%) of biopsies from this population of 143 males/141 females (average age=36.8years; mean BMI=26.6) were completely normal. The remainder showed steatosis (n=107; 37%), steatohepatitis (n=44; 15%), or unexplained low-grade/early stage chronic hepa‐ titis, primary biliary cirrhosis, or nodular regenerative hyperplasia (n=16; 6%). Biopsy find‐ ings disqualified 29/56 donors, negative factors were: obesity, age and liver iron content, contributing to NAFLD pathogenesis. The conclusion was that liver biopsy provides valuable

periportal mononuclear infiltrate, or even piecemeal necrosis, may be seen.

information about otherwise undetectable liver disease in potential liver donors.

About 90 per cent of morbidly obese patients show histological abnormalities of the liver. Morbid obesity may lead to severe disease showing steatosis, ballooning degeneration, lobular inflammation and fibrosis in the study of liver biopsy. These features are similar to the lesions observed in alcoholic hepatitis and may end in cirrhosis and liver failure. Many factors such as, alcohol, drugs, diabetes, viruses, can contribute to progressive liver damage. The development of severe fatty liver disease may be asymptomatic showing a poor correlation with liver function tests. It has been reported that after bypass surgery, weight loss is accompanied by improvement in fatty change and the liver function tests

Histopathologic findings in the liver of 160 patients who were undergoing laparoscopic gas‐ tric bypass or gastric banding for morbid obesity, were recorded, also clinical data (gender, age, BMI and associated diseases) and laboratory evaluation were obtained [108].The diag‐ nosis obtained were : 63 non-nonalcoholic steatohepatitis (non-NASH), 54 NASH, 26 chronic hepatitis B (CHB), 15 alcoholic steatohepatitis and NASH, and 2 chronic hepatitis C (CHC).The coexistence of clinical and histological features of steatohepatitis with another chronic liver disease may reflect the biological significance of the chronic inflammatory con‐

dition in the obese population, which requires further investigation.

*7.10.3. Liver transplant living donor*

58 Liver Biopsy – Indications, Procedures, Results

*7.10.4. Morbid obesity*

are normal.

Another noninvasive method for the assessment of liver fibrosis is elastography (Fibro‐ Scan) [113].

FibroScan device (EchoSens, Paris, France) uses a mild-amplitude, low frequency vibration transmitted through the liver. It induces an elastic shear wave that is detected by pulse-echo ultrasonography as the wave propagates through the organ. The velocity of the wave corre‐ lates with tissue stiffness which correlates well with the degree of fibrosis.

This device is in widespread use in many parts of the world, but is not yet approved in the United States.

Most of the studies have been conducted on patients with chronic hepatitis C but a few studies have also covered fibrosis and cirrhosis due to other etiologies.

This technique, however, has its limitations: it uses expensive equipment, and has decreased accuracy in obese patients and in patients with ascites. Elastography results are not valid in presence of hepatic steatosis, cholestasis, necroinflammation, or portal hypertension. The patient's age and levels of aminotransferases need to be taken into account when interpret‐ ing results of liver stiffness [114].

Fibrotest, and elastography (Fibroscan) as first line estimates of fibrosis in patients with chronic hepatitis C are recommended and liver biopsy will probably be indicated only as a second line diagnosis and reserved for cases of discordance or non-interpretability [112].

Some authors conclude that elastography appears reliable to detect significant fibrosis and cirrhosis in patients with chronic hepatitis C, besides it may turn out to be a valua‐ ble diagnostic procedure and follow-up of patients with chronic liver diseases of differ‐

It is crucial that biopsy interpretation be done by experienced liver pathologists. Pathol‐ ogists have tried to define the features (including length and number of complete por‐ tal tracts) of an adequate liver biopsy sample able to correctly assess the classification of liver fibrosis. Some authors have recommended big samples of 1 to 4 cm in length containing at least 11 complete portal tracts, which could be more reliable for adequate

**Feature Effect on response to therapy Effect on natural history**

Fibrosis Reduces response Presence implies progression

Inflammation No effect Related to increased amount of current

Steatosis Reduces response Related to increased amount of current

Iron accumulation Unclear effect Related to increased amount of current

**Table 6.** Relevance of histological features of chronic hepatitis C to disease progression and therapeutic response

Many intraobserver and interobserver variations have been estimated in the assessment of features, classification, and scoring of liver biopsy assessment. One study reported dif‐ ferences in the evaluation of liver biopsies in chronic viral hepatitis C among 10 patholo‐ gists specializing in liver diseases. These pathologists independently reviewed 30 liver biopsy specimens of viral hepatitis C and completed a histological form for each of the specimens. Five pairs of pathologists were then randomly designated and they inde‐ pendently reviewed the biopsy specimens and filled out a new coding form. The inter‐ observer variation was calculated for each item among the 10 individuals and then

fibrosis and increased rate of

fibrosis, unclear effect on progression

fibrosis, unclear effect on prospective

progression

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61

rate

ent causes [115].

grading and staging [116, 117]

(adapt from Semin Liver Dis 2005)

**8.2. Liver biopsy: Consensus among pathologists?**


**Table 5.** Indication for liver biopsy in different chronic liver diseases in the era of non-invasive methods

In spite of that, elastography is complementary as the combination of noninvasive markers and elastography improves the overall accuracy. In one of the metanalysis, for significant fibrosis, the area under the ROC for Fibrotest and FibroScan were 0.81 (95% CI 0.78-84) and 0.83 (0.03-1.00), respectively [115].

Fibrotest, and elastography (Fibroscan) as first line estimates of fibrosis in patients with chronic hepatitis C are recommended and liver biopsy will probably be indicated only as a second line diagnosis and reserved for cases of discordance or non-interpretability [112].

Some authors conclude that elastography appears reliable to detect significant fibrosis and cirrhosis in patients with chronic hepatitis C, besides it may turn out to be a valua‐ ble diagnostic procedure and follow-up of patients with chronic liver diseases of differ‐ ent causes [115].

#### **8.2. Liver biopsy: Consensus among pathologists?**

This technique, however, has its limitations: it uses expensive equipment, and has decreased accuracy in obese patients and in patients with ascites. Elastography results are not valid in presence of hepatic steatosis, cholestasis, necroinflammation, or portal hypertension. The patient's age and levels of aminotransferases need to be taken into account when interpret‐

**IN FAVOUR of LIVER BIOPSY AGAINST OF LIVER BIOPSY**

Not necessary for diagnosis. Possible use of non-invasive methods in follow-

Not necessary for diagnosis. Possible use of non-invasive methods in follow-

Assessment of fibrosis possible with

Assessment of fibrosis possible with non-invasive methods,( in abstinent

Non validated methods yet for noninvasive assessment of fibrosis

Possible non-invasive assessment of

Non validated methods for noninvasive assessment of fibrosis

Non other options

non-invasive methods

up controls

up controls

patients)

fibrosis

ing results of liver stiffness [114].

60 Liver Biopsy – Indications, Procedures, Results

Chronic HCV hepatitis In selected indications for grading and staging

Chronic HBV hepatitis Grading and staging advisable before

Non-alcoholic steatohepatitis (NASH) NASH is a always an histopathological

Alcoholic steatohepatitis (ASH) ASH is a histopathological diagnosis.

Autoimmune Hepatitis For diagnosis and staging liver biopsy is needed

Primary Biliary Cirrhosis (PBC) Not needed in typical mild cases

Hemochromatosis In general, liver biopsy performed for

Wilson disease For diagnosis and staging and copper

0.83 (0.03-1.00), respectively [115].

diagnosis

starting treatment

But in alcoholic acute hepatitis liver biopsy usually is not performed

without biliary duct damage

diagnosis and staging, and iron

content in the liver

content in the liver

**Table 5.** Indication for liver biopsy in different chronic liver diseases in the era of non-invasive methods

In spite of that, elastography is complementary as the combination of noninvasive markers and elastography improves the overall accuracy. In one of the metanalysis, for significant fibrosis, the area under the ROC for Fibrotest and FibroScan were 0.81 (95% CI 0.78-84) and

It is crucial that biopsy interpretation be done by experienced liver pathologists. Pathol‐ ogists have tried to define the features (including length and number of complete por‐ tal tracts) of an adequate liver biopsy sample able to correctly assess the classification of liver fibrosis. Some authors have recommended big samples of 1 to 4 cm in length containing at least 11 complete portal tracts, which could be more reliable for adequate grading and staging [116, 117]


**Table 6.** Relevance of histological features of chronic hepatitis C to disease progression and therapeutic response

Many intraobserver and interobserver variations have been estimated in the assessment of features, classification, and scoring of liver biopsy assessment. One study reported dif‐ ferences in the evaluation of liver biopsies in chronic viral hepatitis C among 10 patholo‐ gists specializing in liver diseases. These pathologists independently reviewed 30 liver biopsy specimens of viral hepatitis C and completed a histological form for each of the specimens. Five pairs of pathologists were then randomly designated and they inde‐ pendently reviewed the biopsy specimens and filled out a new coding form. The inter‐ observer variation was calculated for each item among the 10 individuals and then among the five pairs. Five features showed an almost perfect or a substantial degree of concordance among the 10 observers (cirrhosis, fibrosis, fibrosis grading by Knodell in‐ dex, steatosis and portal lymphoid aggregates). The 17 other indicators showed a weak‐ er concordance. Five items had a higher concordance when viewed by a pair of pathologists than when studied by only one pathologist (steatosis, periportal necrosis grading by Knodell index, lobular necrosis grading by Knodell index, centrilobular fibro‐ sis, and ductular proliferation). This study reveals that certain features of major impor‐ tance in assessing disease activity show significant observer variation. The acceptable degree of concordance was related mainly to the fibrosis score, whereas other numerical items displayed substantial observer variations. Simultaneous observation by two pathol‐ ogists increased the reproducibility of numerical scoring and of certain viral hepatitis C lesions. A classification of chronic hepatitis C based on dissociated semiquantitative as‐ sessment of necroinflammatory lesions and fibrosis offers more reproducibility than the use of a global numerical index [107].

Ethics related to liver biopsy mainly include issues on the indications, information on poten‐ tial risks and benefits and validity of available alternative options. Patients should be ade‐ quately informed and participate in the decisions for liver biopsy and treatment [120].

> - Well established staging system - Assessment of architectural disturbances related to liver fibrosis - Evaluation of associated lesions (inflammation, steatosis, iron, alcohol)



**Table 7.** Use of liver biopsy in clinical practice. Respective advantages and disadvantages of liver biopsy and transient

What will be the real impact of Liver Biopsy now and in the near future in the era of person‐

**1.** The practice of liver biopsy will remain as an important component in the evaluation of liver disease. However, the value of liver biopsy should be contemplated as a comple‐ mentary tool in modern medicine because of the presence of new non-invasive diagnos‐ tic measures, better prognostication methods and more advances in imaging

**2.** Non invasive tests such as Fibroscan, or similar, adding serum markers will be increas‐ ingly used to identify the amount of fibrosis, and will spare, in most patients, the per‐

**3.** Liver biopsy provides information that is used in conjunction with other data to inform and to guide therapy. The team that joins pathologists, clinicians, radiologists and other specialists meets in order to make clinico-pathological correlations. New classifications

incorporating clinical data in the histological dictamen will be implemented.


Advantages - Direct measure of liver fibrosis

Disadvantages - Invasive and painful

elastography for assessing fibrosis in chronic liver disease

(adapt L Castera & M Pinzani, Gut 2010)

**12. Conclusions**

alized medicine?

techniques.

formance of a liver biopsy.

**Liver biopsy Transient elastography**


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cirrhosis


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congestive heart failure

As a single percutaneous liver biopsy yields only a minute percentage (1 ⁄ 50 000 or 0.002%) of the total hepatic tissue, paired biopsies have been evaluated in several published studies, especially for assessing steatosis and NASH. For quantification, better references are re‐ quired, such as imaging techniques or morphometry, which determines the area of steatosis on liver biopsy.

In fact, as liver steatosis is not homogeneous, classical optical examination of a liver biopsy by a pathologist for measuring liver steatosis by the determination of the percentage of hep‐ atocytes containing lipid vesicles is highly subjective, and steatosis grading corresponds on‐ ly to a semiquantitative scale [68].

The role of the liver biopsy in disease management is evolving nowadays and has to be re‐ considered given the modern pathologic assessment of liver biopsy. Pathologists have made progress in the interpretation of liver biopsies and in processing the information in a concise and scientific way available to clinicians. After evaluating the disease state and interpreting the tests results, the clinician in charge of the patient should consider the individual patient when making recommendations with regards to treatment.

#### **Role of the liver biopsy in personalized medicine**

The liver biopsy specimen aims to obtain a valuable material for the assessment of fibrosis and cirrhosis. Despite limitations related to sampling and interpretation, histological exami‐ nation remains the best standard for staging and diagnosing chronic liver diseases. Its indi‐ cations are decreasing because new therapeutical options for chronic viral hepatitis have improved [118]. Moreover, new non-invasive tests have been developed and their use may increase in the future, especially for long term management [119] (Table 7).

All invasive procedures involve risks, consequently the benefits of obtaining liver for histol‐ ogy should always be weighed against the possible morbidity of the procedure. The deci‐ sion to indicate a liver biopsy has to be taken depending on the center's facilities and the availability of experienced liver pathologists to interpret the biopsy.

Ethics related to liver biopsy mainly include issues on the indications, information on poten‐ tial risks and benefits and validity of available alternative options. Patients should be ade‐ quately informed and participate in the decisions for liver biopsy and treatment [120].


**Table 7.** Use of liver biopsy in clinical practice. Respective advantages and disadvantages of liver biopsy and transient elastography for assessing fibrosis in chronic liver disease
