Many types of direct serum markers that can reflect the degree of fibrosis have been reported. In this chapter, we will introduce four common direct serum markers: hyaluronic acid (HA), laminin (LN), IV type collagen (CIV), and procollagen type III (PCIII).

HA is a type of macromolecule, a glucosamine polysaccharide, that is widely present in the extracellular matrix. It is synthesized by the liver mesenchymal cells and taken up and degraded by endothelial cells. The serum HA level can reflect the status of liver cell damage and liver fibrosis, and it is a sensitive indicator of liver fibrosis and cirrhosis. An elevated serum HA level can indicate possible liver fibrosis. If the serum HA is progressively increasing, it may indicate that liver fibrosis is uncontrolled [15]. LN is a type of non-collagenous structural protein in the extracellular matrix, and CIV is a fibrous glycoprotein. Both proteins are important components of the basement membrane, and they are mainly distributed in the vessel wall, the bile duct and lymphatic walls, and in other places. LN is mainly synthesized by endothelial cells, stem cells, and fat-storing cells in the liver. When liver cells are damaged, LN and CIV combine to form an endothelial basement membrane, resulting in liver fibrosis. Therefore, the serum LN and CIV levels are two important indicators that indicate liver fibrosis in patients with chronic hepatitis [16, 17]. PCIII is the precursor of the III collagen, and it is mainly synthesized and released in the activated hepatic stellate cells. The serum PCIII level can indicate the condition of III collagen metabolism and severity of liver fibrosis, and serum PCIII level is hardly affected by inflammation [18].

Currently, no single direct serum marker can completely independently represent the synthesis of an extracellular matrix. At different stages in the development of liver fibrosis, various serum markers show different trends, and they can be affected by liver inflammation, liver cancer, and other factors, which cause the results to be nonspecific. When inflammation of the liver cells is at the active stage, a large number of extracellular matrices are formed and decomposed, so the direct serum markers may be abnormally high. However, at an advanced stage of liver fibrosis, the activity of the inflammation of the liver is low, so the direct serum marker levels might be inconsistent with the condition of liver fibrosis as observed by pathology. At the same time, when other organs such as the lungs and kidneys are fibrotic, these indicators may also appear elevated, so these markers lack specificity for diagnosing liver fibrosis. In addition, the serum concentrations of these markers are susceptible to impact by the renal excretion clearance.

When liver cells are necrotic to a certain extent, the variables measured by routine blood tests, the coagulation function test, and the liver function test, such as serum transaminase levels, platelet count, coagulation factors, and serum albumin concentration, may change. These indicators reflect the changes of the function of synthesis, metabolism, and reservation in hepatic cells. These simple markers do not directly indicate the production of the liver cell extracellular matrix. Several research groups in China and in Western countries have combined the serum direct or indirect markers to create a model to predict the status of liver fibrosis. Some models even include age or sex. These models are shown in Table 5.2.

Most of the noninvasive models in Table 5.2 are from the United States and Europe, and the population cohorts included are not the same. The main population cohorts in these models are patients with chronic hepatitis C (HCV) infection or patients who abuse alcohol. Most liver fibrosis in Chinese patients is caused by chronic hepatitis B (HBV) infection. The HBV infection rate in the Chinese population cohort is much higher than that in America and Europe. APRI and FIB-4 are two models that have been validated in HBV patients. The initial population cohorts for these two models are HCV and HCV/HIV-infected subjects. Studies have shown that the AUC values of APRI for diagnosing mild or moderate liver fibrosis and cirrhosis were 0.74, 0.73, and 0.73, respectively, and the AUC values of FIB-4 for diagnosing mild or moderate liver fibrosis and cirrhosis were 0.78, 0.82, and 0.84, respectively, in adult patients with chronic HBV infection [19].

Our medical center staffs collected 2176 cases of chronic HBV-induced hepatocellular carcinoma (including 1682 retrospective subjects and 494prospective subjects). All of these patients have had partial liver resection. We found that the AUC values of APRI and FIB-4 for predicting mild or moderate liver fibrosis and cirrhosis in this population cohort are approximately 0.65.By analyzing the data from our medical center, we found that five simple biomarkers were correlated with severity of liver fibrosis: total bilirubin (TBL), platelet count (PLT), clotting time (PT), fibrinogen (FIB), and serum hepatitis B virus e antigen. After univariate and logistic regression analysis, we have established a new model for the assessment of liver fibrosis (not yet published): {[TBL(μmol/L)*PT(s)]/[PLT(10⁹/L)*FIB(g/L)]}*[HBeAg(+) = 2,HBeAg(-) = 1]. The results demonstrated that the AUC of this model for distinguishing early fibrosis (Ishak Score: 0–4) and cirrhosis (Ishak Score: 5–6) was 0.75.

However, because these models combine a variety of markers to assess liver fibrosis, any false-positive marker will affect the diagnostic accuracy. At the same time, the existing serum markers lack specificity for the liver and can be affected by the kidney excretion function. Additionally, various liver or systemic diseases can cause false-positive results for these markers. Although these models have an ideal high accuracy for distinct mild liver fibrosis and cirrhosis, they are limited for distinguishing different levels of moderate liver fibrosis. Direct and indirect serum markers are simple, noninvasive assessment methods for assessing the severity of liver fibrosis. However, the diagnostic accuracy of these models must be studied and validated. We anticipate the findings for simple blood markers that can accurately reflect the severity of liver fibrosis with various etiologies.

For diagnosing liver disease, no serum marker can replace radiographic examination. Radiographic examinations are commonly used noninvasive methods for the diagnosis of liver fibrosis. Combining serum markers with imaging might improve the diagnostic accuracy of liver fibrosis and allow for monitoring dynamically.

When liver cirrhosis reaches a certain level, the two-dimensional ultrasound image can appear as an uneven echo, showing rough or nodular changes of the liver parenchyma; the liver capsule can also be irregular or wavy, and the liver edge is blunt. Abnormality of other organs is also evident, such as thinness and narrowness in the intrahepatic vein, gallbladder wall thickening, and splenomegaly. Recently, general high-frequency ultrasound has been used in the diagnosis of liver fibrosis. High-frequency ultrasound has some value for diagnosing liver fibrosis and early cirrhosis by observing the changes in the surface morphology of the liver capsule and semiquantitative grading. The ultrasonic tissue characterization method uses a radiofrequency or videographic method to explore the relationship between the acoustic characteristics and ultrasonography. The ultrasonic tissue characterization method provides new quantitative indicators for the clinical diagnosis of liver fibrosis.

Based on the two-dimensional ultrasound color, Doppler ultrasound uses the Doppler principle of sound and a series of electronic techniques to show a real-time display of the blood flow spectrum of arteries and veins at a point. Spectral Doppler ultrasound uses the information from the ultrasound Doppler effect to detect the speed of blood flow to diagnose disease. When the liver fibrosis or cirrhosis occurs, sclerosis, an abnormality of the liver parenchyma, damages the integrity of blood vessel walls. Color Doppler and spectral Doppler ultrasound examinations can diagnose liver fibrosis and cirrhosis by detecting the dynamics of the blood in vessels in the liver. Hepatic vein spectra include three types: type 0 hepatic vein – a three- or four-phase wave (i.e., two negative phase waves, one or two positive phase waves); type 1 hepatic vein – a low and flat wave, without inverted blood flow; and type 2 hepatic vein – continuous flat wave, similar to the blood flow in the portal vein [48]. Some researchers believe that normal or mild liver fibrosis is characterized by a type 0 hepatic vein, moderate hepatic fibrosis has a type 1hepatic vein, and severe liver fibrosis has a type 2 hepatic vein. However, the hemodynamic changes of color Doppler and spectral Doppler ultrasound examinations are vulnerable to interference from electronic equipment or human error that will bias the results [49].

Liver contrast-enhanced ultrasound examination is a new method for diagnosing liver disease. The contrast media are gas-filled microbubbles that are administered intravenously to the systemic circulation. The microbubbles sequentially flow through heart, lung, liver artery and the portal vein, the liver sinusoid, and then merge into the hepatic vein. After the signal is emitted by the ultrasound probe, the contrast agents produce harmonic resonance to form an image. Combining the trigger and the acoustic densitometry imaging techniques produces more accurate ultrasound images. Contrast-enhanced ultrasound can be used to image blood perfusion in organs. We can use contrast-enhanced ultrasound to monitor the blood flow in the liver to diagnose liver fibrosis. For the small arteriovenous shunt and hyperdynamic state of microvessels in liver fibrosis or cirrhosis patients, it provides a theoretical basis for contrast-enhanced ultrasound to assess liver fibrosis. The commonly used indicators of contrast-enhanced ultrasound for diagnosing liver fibrosis include the rate of blood flow through the hepatic vein, the delay rate in the hepatic artery, the blood perfusion strength in the portal vein, and the blood cycle time in the liver. Several studies indicated contrast-enhanced ultrasound can reflect the degree of liver fibrosis. It is one of the noninvasive methods to diagnose liver fibrosis and cirrhosis [50, 51].

Three-dimensional ultrasound imaging technology is an emerging discipline in medical imaging. Initial research on three-dimensional ultrasound imaging began in 1970s and now it has entered the stage of clinical application. Its working principle is to create a three-dimensional image via computer using a series of images acquired under certain rules. Three-dimensional ultrasound can show the blood vessels and their positional relationships more clearly than two-dimensional ultrasound for the diagnosis of liver disease. Studies have shown that three-dimensional ultrasound imaging is superior to the two-dimensional ultrasound imaging in many aspects, such as liver morphology, edge of the liver, resolution, continuity of the intrahepatic vessels, and relationship between the liver and blood vessels around the liver [52].

Ultrasound elastography is a new noninvasive diagnostic method for the assessment of liver fibrosis. Currently, there are three main methods used in clinical ultrasound elastography, including the following: real-time tissue elastograph (RTE), transient elastography (FibroScan, FS), and acoustic radiation force impulse (ARFI). The physical principles of these three diagnostic methods for assessing liver fibrosis are different. FS and ARFI base on shear wave elastography; however, RTE uses resilience-tissue elasticity to form images. The modulus of liver elasticity is liver stiffness, which is closely related to its pathological state. Several researchers confirmed the close relationship between liver tissue elasticity and the stages of liver fibrosis [53–55]. The ultrasound elastography technique, with its noninvasive and real-time detection of the soft tissue elasticity modulus, provides a new method for the detection of liver fibrosis and cirrhosis. The new technique avoids the significant risks of liver biopsy to assess liver fibrosis, and it is expected to become the ideal method for evaluating liver fibrosis. However, this new diagnostic method is currently in the research stage, and it has not been widely used in clinical practice. Additionally, its diagnostic accuracy for liver fibrosis caused by chronic HBV infection, alcohol abuse, and liver bile obstructive disease must be validated. Moreover, ultrasound elastography is limited to the identification of different stages of mild to moderate liver fibrosis. Furthermore, the cutoff values of the elastic modulus for predicting different fibrosis levels have not been unified. Therefore, more research is necessary to verify the diagnostic capabilities of ultrasound elastography to fully utilize its diagnostic capabilities.