Liver diseases and their diagnostics

1. Introduction

In the 21st century the technical progress is developing so rapidly. New diagnostic machines appear all over the world and are considered the newest and best tools. Nowadays, the question for whom those devices are the best for, should be asked in a more critical way – best for the patient or best for obtaining a diagnosis. Within the aging of the population, more fatal diseases are diagnosed early and if properly treated, barely reduce the quality of life, while prolonging it.

Yearly millions of people die due to the evolution of chronic liver diseases. The development and accretion of hepatic cirrhosis are strongly connected to the disease progression and to its complications. Therefore, its early detection is essential for the prognosis and guidance of the patients. Noninvasive diagnostic tools are considered a safe, cheap and fast method to investigate a patient without harming him or her. Nowadays, the only alternative is the liver biopsy, which is still considered as golden standard, but it includes minor complications (infec./Bleeding) . The biopsy is expensive, slow and lacks in repeatability in case of measurement errors.

Apart from that, the specimen includes only a very small part of the liver, whereas the histological readings give semi – quantitative information (scale). Up to this day, the permitted noninvasive diagnostics for diffuse chronic hepatopathies is limited on: Serologic Blood Testing, FibroScan, ARFI, SSI, and MR Elastography. In 2003, the first clinically approved system was introduced to the field of research in ultrasound elastography, in order to investigate the significance of organ stiffness.

Liver elastography started with the development of strain elastography (SE), a procedure by which external force – using either the transducer or the patient’s diaphragm – is necessary to measure tissue changes. The idea was to use a color-coded map to display the elastogram in real-time. The shear wave elastography (SWE) uses a mechanical force or an acoustic radiation force impulse (ARFI) to generate shear waves – perpendicular waves – inside the examined tissue and measures the shear wave speed (SWS). The Toshiba Aplio 500 is one of many devices that use the shear wave technology to determine organ stiffness.

This essay is structured in two parts: theoretical part and practical part. The theoretical part includes the diagnostic background knowledge – anatomy and physiology of the liver and furthermore, the pathophysiology of the different types of chronic hepatopathies. This will be followed by a brief overview

of different types of elastography methods. The practical part subscribes the evaluation of a database, with values collected by using the Toshiba Aplio 500 in collaboration with the Department of Gastroenterology and Hepatology, from Victor Babes University of Medicine and Pharmacy, Timisoara, Romania.

This thesis should evaluate if the above-mentioned device is trustful enough to obtain a diagnosis. Moreover, it should demonstrate the importance of the number of measurements as well as the correlation between BMI and LSM. Therefore, 46 patients with liver cirrhosis have been scanned and compared with 43 healthy patients.

2. Liver anatomy and physiology

The liver is the largest and the central metabolic organ of the human body. It is situated in the right epigastric region and is covered by the rib cage. The healthy liver is not palpable and its weight is around 1800 g (♂) / 1600 g (♀). Anatomically it is divided into a right and left lobe but those two can be subdivided into 4 lobes that are considered as functional subunits. Lobus dexter, lobus sinister, lobus quadratus (ventral) and lobus caudatus (dorsal) form the units of the liver. They are subdivided into eight segments, which are separated by fissures and connective tissue ligaments.

Of significant importance for the liver elastography measurements are segments V and VIII, which are situated anteriorly in the right lobe, which are investigated using an intercostal approach. On the lower surface of the liver is the visceral surface, which encloses the hepatic portal.  It includes the hepatic portal vein, proper hepatic artery together with the common hepatic duct. The structure of the liver is determined by its double vascular supply, the biliary ducts and the liver cell trabeculae around the sinusoids. The systemic circulation – hepatic artery – includes only 25% of the blood supply, whereas 75% comes from the portal vein – confluence of superior mesenteric vein and lienal vein carrying nutrient-rich but low-oxygen blood.

Due to its diverse functions, the liver has a differentiated structure. Structural subunit is the liver lobule, which has a polygonal cross section. The corners of the polygon form the portal fields with the Glisson trias, consisting of a branch of the portal vein, the bile duct and a branch of the hepatica artery. Cells of the liver parenchyma include: hepatocytes,  sinus endothelial cells,  kupffer cells, hepatic stellate cells and PIT Cells. The organ itself is the biggest gland and the main metabolic organ to maintain and assure homeostasis. Daily, it produces arround 700 ml of bile which helps in fat digestion and elimination of bile-required substances (bilirubin, medications).

The liver’s physiology contributes to the energy metabolism by glycolysis as well as gluconeogenesis to keep the blood sugar level on normal values. The fat metabolism involves the synthesis of triglycerides, , various as well as the degeneration of fatty acids. Hepatocytes are responsible for the AA and protein metabolism, particularly for the uptake and redistribution of AA, the conversion of AA in glucose and for the binding of nitrogen and ammonia in urea.

Throught hepatic enzymes water-insoluble substances (steroids, medications) are oxidized and conjugated to become water soluble. This detoxicating way of conversion is named biotransformation. The bilirubin metabolism uses this process to eliminate the decay product of old erythrocytes. First the bilirubin is albumin bound transported and then intracellularly by glucuronidation metabolized. The conjugated bilirubin enters the intestine via the gall, where it is further degraded. (1)

In addition to these metabolic functions the liver stores vitamins, in particular: vitamin D  – to prevent a deficiency for about 4 months -, vitamin A – sufficient for approximately 10 months – and vitamin B12 – for at least 12 months.  Moreover, it contributes to forming clotting factors, namely fibrinogen , accelerator globulin, prothrombin and factor VII. The last task consists of the hormone metabolism, which leads to an accumulation of estrogen, aldosteron and cortisol in times of liver damage. (2) Any functional impairment of these liver functions can be the beginning of a lethal sickness.

2.2. Chronic Hepatitis

CH is represented by a list of disorders charcterized by a chronic inflammatory and fibrotic hepatic process in the liver for at least 6 months. In term of differential diagnosis the viral hepatitis has to be distinguished from an autoimmune, genetical or medication induced hepatitis. 60% of the cases are related to virusinduced CH. (HBV, HCV,  HDV) (3)   Every year, 1.4 million people die in consequence of a viral hepatitis and by its related hepatophathies  – liver cirrhosis and Hepatocellcarcinoma, whereas 90% of the deaths are assosciated with HBV and HCV. HAV doesn’t cause a CH.

Characteristic for CH are location  – periportal,  portal and lobular – and the grade of the activity of  the inflammation – mild moderate and severe -, as well as the extended fibrotic stage – none, mild, moderate, severe, cirrhosis. (4) (3) Regions with low prevalence (less than 2%) is Australia, Western Europe and USA.The highest prevalence is in Asia and Africa with over 8 – 10 %. In Romania there is a moderate incidence between 5 -7 %. The WHO has published a report in 2017, whose guidance help to control the outbreak of a viral hepatitis. Vaccination succeded in a 84 % coverage in infancy due to the three dose HBV vaccine.

The key goal should be to prevent most of the infections by vacciantion, early screening and education as they aim for a “road to elimination by 2030” .(5) This essay only deals with the diseases that lead to chronic hepatitis over time namely: HBV, HCV, ALD, LC.

2.2.1. Hepatitis B Virus

The Heptatitis – B infection concerns 5% of the global population and it is most commonly the cause of LC or HCC. Currently, over 350 million people carry the chronic heptatitis virus. The graphic from Lancet 2015 shows that highest HBV incidence in Europ e is in Romania. Abbildung Fehler! Formatvorlage nicht definiert. 1 HBV Incidence Worldwide The virus itself can be  found in blood, saliva and other secretions (breast milk, vaginal and seminal secretions) and can be transmitted horizontally – by blood, blood products, infected instruments, sexual contact or non – sexual physical contact – or vertical – from an infected mother to her child.

In low endemic countries the horizontal transmission is seen more often whereas in poorer countries, vertical transmission is more dominant. (6) HBV is a small size virus originating in the hepadnaviridae family.  HBV belongs to the family of hepatitis DNA viruses. Electromicroscopically, the HBV corresponds to the so-called Dane particle. The HBV consists of a shell, the core, the double strainded DNA and the DNA – polymerase. From the group of the hepatitis viruses, it is the only DNA containing virus. The other hepatitis viruses are RNA viruses.

The incubation time is 30 – 180 days. (3) The action of the liver cell injury is the induction of a cell mediated immume response leading to a destruction of infected hepatocytes. The periportal hepatocyte necrosis with lymphocytic infiltration develops proceeding in a liver fibrosis or HCC at its last stage. (6) For optaining a positive diagnostic of HBV the HBs – AG is the first marker to appear in an AHB and persists after 6 months to confirm CHB. The monitoring of the HBV – DNA indicates the activity of the liver diesase as well as predicting the risk of HCC.

Furthermore, it displays the treatment response and detects a drug resistance. (7) The symptomatic is unspecifc including fatigue, upper abdominal pain, rheumatic symptoms and increased transaminases. The clinical examination might reveal a hepatomegaly with or without a splenomegaly. The liver’s consistency is modified to be stiffer. (6)

2.2.2. Hepatitis C Virus

Chronic hepatitis C is present when HCV- RNA is detectable in the blood for more than 6 months.  The HCV infection is triggered by the RNA virus originating in the Pestiviridae and Flaviviridae families. Its high genetic variability distinguishes between six genotypes and more than 100 subtypes. HCV is diagnosed within 170 million people which corresponds to a 3% of worldwide population.

The socioeconomic indicator also plays a decisive role in the spread of HCV, so the prevalence in Germany is 0.3%, compared to 22% in Egypt. In Romania this prevalence is slightly elevated to 3.5%. Globally, HCV infection causes 30% of cirrhosis and about 25% of primary HCC. (3)(6) The risk group for HCV includes mainly drug addicts, who share unsterile needles and patients who received a blood transfusion or even an organ transplant. Similar to HBV there is a horizontal and a vertical way of transmission.

The incubation time is 15 – 180 days. (3) HCV is a cytopathic virus that causes hepatocyte lesions by direct toxic action and immunologically mediated mechanism. The failure of the liver to eliminate the virus is followed by a chronic lesion of the liver and results in liver cirrhosis. (6) Characteristics are strongly changing transaminase activities (1.5 up to 10 times elevated) and serological detection of Anti-HCV-AB and HCV – RNA play a decisive role. The determination of the genotype requires to adjust the method and duration of therapy. In HCV infection the clinical picture is most frequently meaningless and unspecific.

Similar to HBV fatigue with upper abdominal pain combined with a hepatosplenomegaly shows an advanced stage of the disease. New therapeutic guidelines of cronical hepatitis C differentiate between the HCV genotype and the degree of cirrhosis. An 8 to 12 week treatment regime, with new antiviral drugs like direct – acting agents (DAA), shows a sustained HCV elimination or even a cure in 95% of the cases.(8) The aim of the treatment consists of the lack of detection of HCV RNA in the serum 12 weeks after the end of therapy. Then, one can assume a complete cure.(1)

2.2.3. Liver Cirrhosis

In LC, which is the last stage of various chronic liver diseases, there is an irreversible transformation of the liver parenchyma. Extensive fibrosis and diffuse connective tissue proliferation destroys the microscopic structure of the liver lobules resulting in the arise of regeneration nodules. René Laënnec, a French doctor who invented the stethoscope, named the disease after the Greek word “kirrhos” which means yellow – orange, lemon – yellow, referring to the icteric skin changes. This process of remodeling is lasting years or tens of years while the presence of necrotic and inflammatory lesions result in the functional impairment namely hepatic insufficiency and portal hypertension. (6),(1)

LC is the 14th most deadly disease worldwide but fourth in central Europe with an increasing trend. Its 1st year mortality range extends from 1 % to 57% based on stage and cause of disease. (9) Etiologically, 50 – 60 % of all LC cases are alcoholic natured (Laënnec cirrhosis) followed by 15 – 30% viral causes (HBV, HCV and HDV). The remaining causes of LC are cholestatic -, metabolic -, vascular, drug -, nutritional causes as well as autoimmune – and cryptogenic cirrhosis. (6),(1) For the macroscopic differentiation the liver morphology changes are measured by the size of the regeneration nodules to distinguish micronodular (usually alcoholic, 2-3 mm in all lobes) from macronodular (postviral, > 3mm). The mixed micro – macronodular form is found in biliary cirrhosis.

The patient’s clinic depicts general symptoms, liver skin signs, hormonal disorders and complications of the liver decompensation. Prior to the introduction of transient elastography, the Child Pugh Classification was used to differentiate the severity of LC. The one-year survival rate in Child A is 100% unlike in Child C with 35%.  The therapeutic goal is to avoid hepatotoxic substances and the treatment of the underlying disease with its complications. The only cure is a liver transplantation. (3)(6)(1)

2.2.4. Alcoholic Liver Disease (ALD)

Profuse alcohol consumption is a healthcare problem all over the world. In 2012, 3.3 million people died, as a consequence of daily alcohol intake over the toxic limit of 40 – 80 ml absolute alcohol/day for men and 40 – 50 ml for women, according to WHO 2014. Chronic alcohol abuse, characterized by a variety of structural and functional changes, is related to a significant increase in mortality and morbidity. (6) (10) (11)

Histologically ALD is present in three forms: alcoholic liver steatosis (ALS), alcoholic hepatitis (AH) and chronic hepatitis including fibrosis and cirrhosis. ALS is diagnosed when more than 50 % of the liver parenchyma is greasy, which can be cured by the cessation of alcohol consumption. It progresses into the AH, which might be asymptomatic or which might show signs of inflammation and a more significant clinical picture including abdominal pain, weight loss, fever, icterus, ascites, hepatomegaly and possibly hepatic encephalopathy.

Specific for the AH is the appearance of Mallory bodies (alcoholic hyaline) neutrophilic inflammatory infiltrate, necrosis, fibrosis and the ballooning degeneration. (1)(6) Therapeutical corticosteroids, especially the application of prednisolone are prescribed in order to suppress the immune response from the moderate to the severe forms of AH. Nonetheless, abstinence of alcohol is of capital importance to prevent liver tissue damage. (10) Ultrasound can help in the non – invasive diagnosis of the ALD showing a greasy alteration of the liver parenchyma as well as morphological signs of the liver cirrhosis. The worst disease progression would be the development of a HCC with its known consequences. (12)

2.2.5. Non Alcoholic Steatohepatitis (NASH)

The evidence of fatty changes within the liver in a patient, who does not present a history with excessive alcohol consumption, defines NASH. Histologically, the lesions range from a benign biopsy to a hepatic steatosis without inflammation, to steatosis hepatica, characterized by inflammation and fibrotic changes all the way to liver cirrhosis. (13) Obesity, Diabetes Mellitus Type II, female gender and hypertriglyceridemia are risk factors and commonly associated with NASH. 70% of the obese (BMI > 30 kg / m2) and 30% of diabetics show sonographically a liver steatosis. (1)

The pathogenesis is insufficiently defined but the key role plays an increased hepatocyte fatty acid uptake combined with an enhanced insulin resistance. (14) (6) The clinical picture of NASH patients is mostly asymptomatic. Unspecific symptoms include fatigue, malaise and discomfort in the right hypochondrium. In advanced disease cirrhotic signs in form of jaundice and ascites may appear. Often, it is an incidental finding associated with an US examination. Imaging (US, CT), liver biopsy (Golden Standard) and serological testing (excluding DD) confirm the diagnosis.

The hepatic fat shows an increased echogenicity in US; while it appears hypodense in contrast free CT scan. The LB is a powerful tool to exclude first of all other pathologies as well as on the other hand to permit a staging of patients with NASH. (13) The therapy of NASH is based on life style changes, namely: weight loss, diet and exercise. Bariatric surgery appears to be a promising therapeutic procedure as it leads to an improvement in insulin resistance and for this reason initiates a decrease in fatty liver. The prognosis of NASH is favorable, but may also lead to LC and HCC in rare cases. (15)(1)

3. Ultrasound Elastography

Since the introduction of ultrasound in the 70s into the clinical practice, it has been renewed and improved again and again. Three technical completely different developments lead to a better clinical examination and thus, to a more appropriate therapy. First, B-Mode displays anatomy using acoustic impedance, while Doppler measures motion to display vascular flow. Thirdly, there is elastography –  referred to as “palpation imaging”- measuring mechanical properties to display tissue stiffness.  (16)(17)

Elastography is a non-invasive science that evaluates organ stiffness using an external force or vibration to stress the organ. Modern ultrasound devices are capable of measuring the organ deformation and calculate a ratio. Qualitative and quantitative techniques have been developed. SE and SRI use a quasi-static type of force to display a strain rate to obtain a qualitative diagnostically conclusive degree of stiffness, while ARFI, TE, pSWE and SWE utilize a dynamic type of force to display organ deformability within a ROI as a quantitative elasticity image (elastogram).

Stiffness correlates with pathological changes that makes it relevant from a diagnostical point of view. A plethora of different approaches has been developed, with advantages and disadvantages on each of the different methods. Several studies have shown that elastography is improving the diagnostic accuracy. Tumors of the breast, the prostate, the cervix, and the thyroid gland had been successfully evaluated and benign and malignant lymph nodes were differentiated with an accuracy between 85% – 90%. (18)

3.1. Transient Elastography (TE)

Transient Elastography (TE) is a non-invasive ultrasonic method using FibroScan, (Echosens, France) which is based on the principles of Hooke’s law, to deform elastic materials. „In an elastic member stress is directly proportional to strain within elastic limit“ σ_ij=C_ijkl ϵ_(kl ) (19) Every stress σ_ij is followed by a material strain ϵ_(kl )whereas C is used as a constant named Young’s modulus of elasticity (E) that adjusts on the type of material and its characteristics.

The force applied to the material is proportional to the extension up until the limit of proportionality where it is still elastic but not linear. E=3ϕv^2 This law mathematically shows Young’s modulus E (kPa), that can be calculated by the multiplication of 3 – as a constant, ϕ – expresses the tissue density (kg/m^3 ) – and v – represents the wave speed (m/s). (20) FibroScan works with vibration controlled transient elastography (VCTE) to create shear waves through vibrations of low frequency (50 MHz) and mild amplitude.

The device uses a pulse – echo ultrasound to track the shear wave and evaluates the SWS to provide a LSM, which correlates with liver fibrosis stage and is expressed in kilopascals (kPa). Due to a high error rate with the M – Probe for obesity and children, three more transducers were developed to adapt to patient morphology and take into account the following parameters: skin – capsule distance (SCD) and thoracic perimeter (TP).

For the LSM the patient should be lying in dorsal decubitus and – to ensure the best echographic window – with the right arm in maximal abduction. Using an intercostal approach the right liver lobe is visualized with an A-Mode image and a six cm thick liver parenchyma, free of biliary structures and large vessels, is required. After finding such a location, the operator presses the probe button to begin the measurement. The software automatically rejects incorrect acquisitions. 3 TE diagnostic volume exceeds the liver biopsy by a 100 times and thereby more significant.

The elasticity of the liver is measured in kPa and contains a range from 2.5 kPa to 75 kPa. 10 measurements should be evaluated with an IQR/M ≤ 30 %.  The XL – Probe lacks in diagnostic accuracy because the Young’s modulus is lower then in those patients measured with an M – Probe. (22) The ultrasound attenuation coefficient (dB/m) is responsible for the CAP. Its values range from 100 to 400 dB/m, and its value is directly proportional to the level of steatosis. (20) Limiting factor for this TE evaluation are patients with ascites, obesity and a narrow intercostal space. (23)

3.2. Acoustic Radiation Force Impulse (ARFI)

The ARF results from a transfer of momentum that originates in an acoustic wave with a barrier along its path, where F (kg/(s^2 〖cm〗^2 )) is the magnitude of the acoustic radiation force, α (Np/cm) represents the acoustic absorption coefficient, I (W/〖cm〗^2 ) expresses the temporal intensity of the acoustic beam, and c (cm/s) shows the speed of sound within the medium. (24) (25) F=2αI/c The force (F) slightly displaces the investigated organ at a focal region according to the law of Hook. In order to detect the displacements the transducer has to switch into imaging mode to track the so-called “sperckle” in order to display stiffness properties.  (17)

This method has two measurable consequences: firstly it can evaluate directly the strain elastography (qualitative); secondly it generates shear waves whose speed can be used for SWE (quantitative) evaluation. (16) ARFI elastography is can be applied using the Siemens AcusonS2000TM (Siemens AG, Erlangen, Germany) ultrasound system. It evaluates the tissue elasticity utilizing a high intensity acoustic “push pulse” to generate shear waves. Propagation of those waves in fibrotic tissue is increased. SWS is measured in an anatomic ROI and is expressed in m/s. (26)(27)

LSM is performed on the patient in left lateral decubitus, the right arm in maximal abduction. In order to avoid cardiac movements, the right liver lobe (Segment VIII or V) is investigated using an intercostal approach. The rectangular ROI is placed about 1 cm below the capsule and should be free from large vessels and bile ducts. The patient is then asked to stop breathing for some seconds in order to obtain a correct result. 10 Measurements are done and their medium value is calculated. (27) Conclusively ARFI is reliable to quantify the level of fibrosis in chronic liver hepathopaties and can help to modify treatment adaptations in order to improve the patient’s prognosis. (28)

3.3. Supersonic Shear Imaging (SSI)

SSI is a new non-invasive ultrasound based technique visualizing in real – time viscoelastic properties. The Aixplorer ® system is utilizing a unique Ultrafast TM technology – with a 200 times faster frame rate compared to conventional US systems – to map a qualitative organ stiffness. Unlike ARFI the transducer sends multiple SuperSonic focused ultrasonic beams into the ROI where they generates shear waves.

The SWS is then evaluated using an ultrafast scanner to measure the Young’s Modulus so that elasticity can be displayed on a color-coded map in which soft tissue is shown in blue and stiff tissue shown in red. (29) (30) The Aixplorer ® system comes with a variety of eleven transducers that are capable of SWE.  For the LSM, a linear transducer is positioned in the intercostal space to evaluate the right lobe of the liver. The patient is in a supine position and has to put the right hand behind his head, so that the arm is in maximal abduction. During the measurements the subject is asked to stop breathing in order to rule out measurement errors.

After obtaining a B-Mode Image, a rectangular area of interest is selected, which is 1 – 2 cm below the Glisson’s capsule and an area free of big vessels. Then shear wave modus is selected inside of the AOI. A circular ROI is then used to display the tissue stiffness. (30)(16)(31) Abbildung Fehler! Formatvorlage nicht definiert. 5 XXX ; SSI Imaging Since 2D – SWE is not working with a single shot but with a continuous method so that SWE acquisition is continued for several seconds.

The newest EFSUMB guideline update from 2017 recommends only 3 measurements, as studies have shown that it is sufficient. The most common failure of the measurement is not picking the ideal depth 4 – 5 cm. Other measurement inaccuracies are favored by the subject’s morphology like large amounts of ascites, pulsatile movements, reverberations or intercostal wall thickness. (22) The Aixplorer ® system provides the option to either evaluate in kPA or in meter per second if its required. (16)

3.4. Time Harmonic Ultrasound Elastography (THED)

THED is based on the vibration phase gradient elastography that was found by Yamakohsi in 1990. This PG sonoelastography is used to reconstruct SWS inside an image and was applied to skeletal muscle. 2014 Tzschätzsch modified this multifrequency excitation to prove SWS values inside the liver. This method was then extended into a 2D quasi – harmonic method: external vibration multidirectional ultrasound shear wave elastography (EVMUSE). Due to a burst of 50 Hz – vibrations disuse displacement is evaluated within a relaxation phase of some milliseconds.

For 2D THE, tissue is excited by a loudspeaker and motion is captured with an ultrasound transducer.  It is currently used to evaluate organ stiffness in liver, spleen and prostate. (19) THED Research System comes with a special medical table which as an integrated loudspeaker on the bottom side. The speaker induces soft low frequency waves into the patient to generate shear wave. The system includes a Sonic 1 Ultrasound Scanner (Analogic) that can be used for standard ultrasound examination as well.  In order to evaluate soft tissue stiffness collected ultrasound data is collected and sent to a second computer that displays on a full field elastogram.

The examination is done on this special patient bed, subject in supine position, with the loudspeaker placed underneath the liver. The patient should fasten at least 2 h before the investigation. The transducer utilizes an intercostal approach to visualize the right liver lobe in B – Mode while avoiding the large vessels inside the liver parenchyma. By pushing a button the evaluation starts. The examination time should not exceed the 5 minutes per patient. Ten measurements are done accomplishing the criteria of SR ≥ 60% and IQR < 30%.

Penetration depth with 13 centimeters extends its application area. The measurement takes 1 second and the resulting color-coded elastogram is displayed on the elastography monitor. It is calculated over the Anti – B mode image resulting in the Full Field View Elastogram. Due to the fact that there are only two of these devices, there is hardly any medical research in this area. THE 2D SWE is a cost efficient tool providing immediately assessment of the liver morphology without the limitations of obesity and ascites. (32)