Expert Review of Medical Devices

ISSN: 1743-4440 (Print) 1745-2422 (Online) Journal homepage: http://www.tandfonline.com/loi/ierd20

Extracorporeal support for patients with acute and acute on chronic liver failure Jonathan Aron, Banwari Agarwal & Andrew Davenport To cite this article: Jonathan Aron, Banwari Agarwal & Andrew Davenport (2016): Extracorporeal support for patients with acute and acute on chronic liver failure, Expert Review of Medical Devices, DOI: 10.1586/17434440.2016.1154455 To link to this article: http://dx.doi.org/10.1586/17434440.2016.1154455

Accepted author version posted online: 19 Feb 2016.

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Date: 22 February 2016, At: 17:55

1 Publisher: Taylor & Francis Journal: Expert Review of Medical Devices DOI: 10.1586/17434440.2016.1154455 Extracorporeal support for patients with acute and acute on chronic liver failure

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Jonathan Aron, Banwari Agarwal and Andrew Davenport Corresponding Author Andrew Davenport [email protected] Royal free Hospital - UCL Centre for Nephrology London United Kingdom of Great Britain and Northern Ireland Abstract The number of patients developing liver failure; acute on chronic liver failure and acute liver failure continues to increase, along with the demand for donor livers for transplantation. As such there is a clinical need to develop effective extracorporeal devices to support patients with acute liver failure or acute-onchronic liver failure to allow time for hepatocyte regeneration, and so avoiding the need for liver transplantation, or to bridge the patient to liver transplantation, and also potentially to provide symptomatic relief for patients with cirrhosis not suitable for transplantation. Currently devices can be divided into those designed to remove toxins, including plasma exchange, high permeability dialyzers and adsorption columns or membranes, coupled with replacement of plasma proteins; albumin dialysis systems; and bioartificial devices which may provide some of the biological functions of the liver. In the future we expect combinations of these devices in clinical practice, due to the developments in bioartificial scaffolds. Key words acute liver failure, acute on chronic liver failure, plasma exchange, adsorption, biological and plasma filtration

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Introduction

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The liver is the largest internal organ in the human body, weighing 1.4-1.6 kg in a healthy human adult and performs more than 500 separate biologic functions amongst which biosynthesis and detoxification are the most important. Liver disease results in decreased production of proteins such as albumin and coagulation factors and failure to eliminate endogenous and exogenous toxins leading to development of coagulopathy and hepatic encephalopathy, and other organ dysfunction. Due to the complexity of its functions, the creation of a liver support system to simulate an artificial liver has been less successful than for other organ support therapies. A number of different strategies have been employed to provide extracorporeal liver support. The simplest model is an extracorporeal haemofilter, similar to that used for renal replacement therapy (RRT), which in addition to removing water soluble solutes, attempts to remove lipophilic, protein bound solutes by using a variety of membranes and adsorbents. More complicated systems attempt to emulate the synthetic, metabolic and regulatory activities of the liver using human or animal cells capable of performing the function of hepatocytes. Systems that combine these two processes also exist to simultaneously provide biosynthesis and detoxification. This chapter will describe the spectrum of liver disease and how an effective support system could potentially add to the current management of these conditions. In addition some of the commonly used devices will be described and their effectiveness discussed. The Liver and its key functions

Hepatocytes play a key role in synthesis and degradation of proteins, including albumin, bile, coagulation factors, complement proteins and amino acids. However the liver also contains endothelial cells and macrophages. Sinusoidal endothelial cells separate blood from hepatocytes and perform important tasks such as molecular transportation, regulation of endogenous waste and inflammatory cell signalling. Kuppfer cells are resident macrophages which remove circulating endotoxin and bacterial. These Kupfer cells represent 80% of the total macrophage population in the body and can mobilize into the circulation as part of the host response to injury. Albumin, the most abundant protein in the body, is a large MW protein (65-70 KDa) produced solely by liver hepatocytes. Both the quantity and the quality of albumin are dependent on liver function. As albumin is of particular importance it is considered separately, later in the article. Apart from Von Willibrand factor (VWF), which is produced by the vascular endothelium, all the other coagulation proteins are made by hepatocytes. Vitamin K-dependent coagulation factors (II, VII, IX, X, protein C, S and Z) are reduced early in liver disease with the exception of fibrinogen, which is often raised as an acute phase protein.

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The liver plays a key role in deamination, in which the nitrogen molecule from amino acid metabolism is converted to ammonia and the non-nitrogenous component is incorporated to synthesise carbohydrates or fats. Ammonia combines with carbon dioxide in peri-portal hepatocytes to form urea and water in a chemical process collectively called the ornithine cycle. Alternatively, condensing ammonia and glutamate forms glutamine. The conversion of glutamine to glutamate is reversible, and this reaction is used to control metabolic acid-base balance, with the generation of urea. The enzyme glutamate synthase is found in both hepatocytes but also astrocytes which regulate the blood brain barrier (BBB). As the liver fails, astrocytes shoulder an increasing burden to convert nitrogenous compounds into glutamine, resulting in astrocyte swelling, disruption of the BBB and encephalopathy. .

Bilirubin is the degradation product of haem and is actively transported into hepatocytes to be conjugated with glucuronic acid and then excreted into gastrointestinal tract. Failure of this conjugation process results in unconjugated hyperbilirubinaemia. Chronic liver disease leads to remodelling of the normal liver architecture causing obstruction of biliary tracts, resulting in a conjugated hyperbilirubinaemia. Drugs and hormones are deactivated and excreted through phase 1 (oxidation, reduction, hydrolysis) and phase 2 (conjugative) reactions. This staged process first creates a polar substance thus allowing the addition of an organic compound. The subsequent compound typically has less pharmacological activity, but increased water-solubility so facilitating renal excretion. Other vital hepatic functions include the synthesis of bile from cholesterol, glucose homeostasis, lactate metabolism, immune defence.

The consequences of liver failure are therefore varied but primarily three manifestations are commonly apparent. The first is the accumulation of endogenous and exogenous toxins that results in encephalopathy and multiorgan failure. The second is the failure of biosynthesis that results in hypoalbuminaemia and coagulopathy. The third is immune paresis and overwhelming sepsis. Aetiology of liver failure Due to great advances in the management of a number of high-profile conditions, particularly heart disease, the mortality rate resulting from liver disorders has increased fourfold in the last 50 years and is now the third most common cause of premature death in the UK (1). Most patients die during their working lives, which has a significant impact on society. In 2012, 60,000 people in England and Wales suffered from liver disease, of which 10% had cirrhosis (2). This led to nearly 60,000 admissions to hospital and over 10,000 deaths (2). The causes of acute and chronic liver disease differ.

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Intentional or inadvertent paracetamol (acetaminophen) toxicity is the most common cause of acute liver failure (ALF) in industrialised countries. The incidence increased from the 1970s to 1998, when the quantity available for purchase was limited to 8g. Around 82 000 to 90 000 patients present with paracetamol overdose in the UK each year, resulting in between 150 to 250 deaths per year. Most of these are attributable to late presentation, a staggered overdose or after unintentional ingestion. Other drugs may induce liver damage either due to a hypersensitivity reaction or due to a prolonged exposure to a hepatotoxin. Ecstasy and cocaine are rare causes of drug-mediated ALF (1,3).

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Acute liver disorders caused by viruses are significant, representing 40-70% of patients worldwide (4). Hepatitis A, B, D, E, herpes simplex, varicella zoster, cytomegalovirus, Ebstein-Barr virus and measles virus are all causes of ALF.

Due to public health initiatives to reduce alcohol consumption, the rate of liver disease in France, Italy and Spain has declined over the last four decades. However, alcohol-induced liver disease is increasingly represented in the UK and presents a significant public health concern. The increased availability and affordability of increasingly strong preparations of alcohol is likely responsible (5). One quarter of the population drink over the recommended limit and these individuals account for three-quarters of the country’s overall consumption. One third of patients with alcohol-related liver disease demonstrate dependence and a third of life-long drinkers will develop cirrhosis (6). Obesity represents another public health problem, with 62% of adults and 28% of children being either overweight or obese according to UK government figures. Accordingly, 25% of the population have demonstrable non-alcoholic fatty liver disease, and 10% have evidence of fibrotic changes. Progression towards cirrhosis and hepatocellular carcinoma primarily due to obesity is increasing (7). Chronic viral hepatitis remains important in western societies, but is more prevalent in the developing world. Current anti-viral treatment may lead to the eradication of hepatitis B and C within 30 years.

Other causes of liver disease include vascular-occlusive disease, ischaemic damage, heart failure, autoimmune disorders, congenital diseases, inflammatory conditions and pregnancy-related complications. Liver failure Syndromes

Liver failure may be classified in terms of the progression of disease ranging from acute, acute-on-chronic and end-stage liver failure (8). In addition, smallfor size syndrome (SFSS) describes a post-operative state following large volume liver resection for malignant conditions or following live related liver transplantation and is discussed below. Acute Liver failure (ALF)

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ALF is characterized by a rapid decline in liver function in a previously healthy liver. This occurs due to massive hepatocyte necrosis following an insult secondary to drug toxicity, infection, pregnancy-related disorders, vascular interruptions, cardiac output failure or metabolic syndromes. The predominant clinical manifestations are failure of bilirubin metabolism resulting in jaundice, the accumulation of toxins resulting in hepatic encephalopathy (HE) and the failure of biosynthesis resulting in coagulopathy. Cerebral oedema and immuneparesis resulting in sepsis are the two most common causes of mortality. Depending on the duration of jaundice prior to encephalopathy, ALF can be further divided into hyper-acute (1-7 days), acute (8-28 days) and sub-acute (28 days - 6 months) to aid prognostication and management. This categorisation, in addition to metabolic markers, aetiology and patient age determine the risk of death and identifies which patients are unlikely to survive without a liver transplant (LT). LT is a life-saving procedure but is associated with significant morbidity and mortality. Acute-on-chronic liver failure (ACLF)

ACLF, previously defined as acute decompensation of chronic liver disease, is now seen as a distinct clinical entity, characterised by mounting of severe systemic inflammation inresponse to a precipitating event, identified in majority of patients, and leading to both liver and extrahepatic organ dysfunction, and associated with hugh short term mortality (ref gines, gastroenterology, ref 2 angeli Jhep). This was a result of formation of a europe wide consortium of scientists in liver ds; CLIF) is an acute deterioration of liver function over a short time in a patient with previous (known or otherwise) well compensated liver disease, associated with a precipitating event such as sepsis, gastrointestinal bleeding or further direct liver injury, leading to further decline in the liver function (acute decompensation) and development of other organ failure which is associated with a high short term mortality. Irrespective of the nature of the precipitating event, the final common pathway responsible for organ dysfunction appears to be related to development of intense dysregulated systemic inflammation. The liver may still have the ability to regenerate and return to baseline functionality if the cause of the acute deterioration can be resolved. The CLIF (Chronic Liver Failure) Consortium, an organisation that promotes research into chronic liver disease, continues to investigate epidemiology and management to improve patient care. End-stage liver disease (ESLD)

ESLD results in the progressive decline of liver function, from the compensated state to the decompensated state. It is characterized by the inability to regenerate and represents the terminal phase of liver failure. The only treatment modality available is liver transplantation. Small for size syndrome (SFSS)

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SFSS is a clinical syndrome that may occur after liver transplantation or after extended hepatectomy. This may occur in a live-donor or recipient. For transplanted organs, this may occur due to the use of split-liver transplants and marginal grafts. It is characterized by post-operative coagulopathy and liver dysfunction due to an insufficient functional liver mass. The remaining functional liver must be greater than 25% in a healthy liver or 40% in a suboptimal liver to function adequately after resection (9).

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Albumin in liver disease

Albumin is normally present in the serum at concentrations of 35 -50 g/l and constitutes 50% of all plasma proteins. 10 – 15g of albumin is produced daily depending on the colloid osmotic pressure surrounding the hepatocytes (10). The majority of albumin escapes into the interstitial fluid, at a rate that is regulated by capillary permeability, oncotic and hydrostatic pressures, eventually returning to the circulation via the lymphatic system. Its half-life is around 15 days and degeneration occurs in the skin, muscle, liver and renal cells at a rate that equals production in health.

The functions of albumin include maintenance of oncotic pressure and acid-base balance. In addition, albumin has many binding sites, serving as a transporter for thyroid and steroid hormones, unconjugated bilirubin, fatty acids and drugs. Of particular importance, albumin carries anti-thrombin and heparin co-factor, responsible for moderating the coagulation cascade. Mercaptoalbumin, albumin with a free redox-active thiol group, is a scavenger for many reactive oxygen and nitrogen species (11). Albumin also has several interactions with the immune system. In animal models, it has been shown to reduce tumour necrosis factor alpha (TNF�) and increase expression of vascular cell adhesion molecule one (VCAM1) and nuclear factor KB(NFκB) (12). Therefore albumin has several important roles in reducing oxidative stress, decreasing inflammation and immune-cell signalling.

Liver disease affects the quantity and quality of albumin by disrupting its synthesis, distribution and breakdown. Hepatocyte dysfunction results in decreased synthesis of albumin and serum albumin is used as a surrogate of function and a marker of disease severity (13). In addition, albumin itself may be dysfunctional, either as a result of over-saturation with bilirubin, or due to changes in its structure. For example, a study demonstrated that albuminbinding capacity was reduced 40% in the presence of hyperbilirubinaemia (14). Another study demonstrated a reduced ability to bind fatty acids in patients with acute on chronic liver failure, which was of prognostic significance (15). Oxidised albumin is more abundant in patients with liver disease and once irreversible damage has occurred, levels correlate with 30 and 90-day mortality (16). Liver disease causing multi-organ dysfunction

Endotoxins, such as lipopolysaccharide (LPS) from gram-negative bacteria and peptidoglycan (PGN) or lipopeptide (LP) from gram-positive bacteria activate

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toll-like receptors (TLR) and initiate cytokine release (17). Under normal circumstances the Kuppfer cells are able to rapidly clear LPS originating from the gastrointestinal tract from portal blood. This process is impaired in liver dysfunction due to immune paresis. Indeed endotoxin levels increase progressively as liver function deteriorates and mortality is higher in these patients (17). Excess cytokine production without modulation can cause overwhelming shock. TLRs are up-regulated and monocytes are activated due to increased cytokine presence but fail to adequately activate the innate immune system. The cause of immune paresis is multi-factorial. Monocyte function is altered and phagocytosis reduced. Kuppfer cell numbers are reduced and the alteration of liver architecture results in bypassing of portal blood leaving some regions of liver unsupported. Complement levels are reduced and opsonisation is less effective. Tissue hypoperfusion occurs as a result of vasoplegia, endothelial dysfunction, low oncotic pressure, systemic hypoperfusion, microvascular dysfunction, and increased micro-thrombi. This leads to an additional ischaemic insult resulting in the production of free radical species which then propagates further hepatocyte damage (18).

Encephalopathy occurs in patients with ALF, ACLF and chronic liver failure (CLF), however the pathophysiology in CLF differs somewhat as it is rarely associated with intracranial hypertension. Ammonia levels are frequently elevated in these patients due to impaired detoxification in the liver (19). Extrahepatic detoxification can occur within astrocytes resident within the BBB but causes cellular dysfunction and contributes to encephalopathy (20). Proinflammatory mediators modulate the response of the brain to ammonia with 70% of patients with bacteraemia demonstrating encephalopathy (20). Amongst these, cytokines such as IL-1, IL-6 and TNF�-a may act on receptors found on the endothelium of the brain resulting in increased uptake of ammonia. Reactive species production increases and the ability to bind and remove these are limited resulting in astrocyte damage (21). Renal failure occurs commonly in the context of ALF, with up to 50% requiring renal support therapy. Hepatorenal syndrome (HRS) is renal failure that does not respond to optimisation of the plasma volume in patients with liver disease. Type 1 HRS occurs in the presence of haemodynamic instability and Type 2 HRS occurs more insidiously often in the presence of ascites (22). The causes are multi-factorial. Acute tubular necrosis due to pre-renal failure is a common finding in both groups of patients. The causative hepatotoxin may exert direct injury to the renal tubules as well, for example in paractemaol overdose. In chronic disease, renal failure develops more frequently in patients with spontaneous bacterial peritonitis (SBP) (23).

Characteristic cardiovascular changes occur in patients with ALF. These patients develop profound vasoplegia that results in a hyperdynamic circulation. These patients often respond to intravenous fluid therapy but may need vasopressor and inotrope support. Cirrhotic cardiomyopathy may also occur and troponin levels are often elevated (24). This manifests with altered contractile

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responsiveness to stress, diastolic dysfunction and reduced chronotropic response. Intractable shock may be due to adrenal failure and may respond to steroid replacement. Patients with cirrhosis demonstrate a similar cardiovascular response which may be complicated by large volumes of fluid in the peritoneal compartment and blood loss from gastric erosions or varicies.

Respiratory failure is common in ALF due to encephalopathy resulting in a failure to protect the airway, hypoventilation and aspiration. Atelectasis and pulmonary vascular changes result in significant ventilation-perfusion mismatching and hypoxia. These patients demonstrate immune-paresis and chest-related infections are common. In patients with cirrhosis, similar changes are evident. Hepatopulmonary syndrome and portopulmonary hypertension may occur in patients with cirrhosis or portal hypertension, which does not occur in patients with ALF. In addition tense ascites can inhibit effective ventilation and pleural effusions may further impair gas exchange. The management of liver failure

The management of liver failure is complex and depends on the presentation. Put simply it can be categorised into 3 stages. The first is the identification of the cause and treatment to reverse or limit the process to prevent organ failure. The second involves supportive therapy once the organ has failed so that recovery of function may occur, if possible. Finally if recovery is no longer possible, then the organ should be replaced (transplantation).

In ALF if the underlying cause is identified and treated appropriately, supportive treatment and time may be sufficient to allow spontaneous resolution. Examples of effective ‘antidotes’ include n-acetyl cysteine (NAC) for paracetamol toxicity or steroid treatment for autoimmune disease. For patients with ACLF, supportive treatment during an acute deterioration whilst the precipitating cause is addressed may result in a return to baseline or near-baseline function. However, liver transplantation currently remains the only definitive treatment option for irreversible ALF or ACLF. The UK has performed over 7000 transplants in the last 10 years and in 2014 performed 911 operations, the majority from brain-stem dead donors (25). Each year the number of patients on the active transplant list increases. Currently the mean wait for an elective adult liver is 144 days (133-155 95% CI) and 2 days for a super-urgent liver (25). There is an on-going reduction in organ availability when compared to organ demand and around 20% of patients on the transplant waiting list will die while waiting for an organ. The need for an extra-corporeal liver support system (ECLS)

In all the conditions listed above, the liver loses the ability to synthesise and detoxify resulting in significant morbidity and mortality. Thus a liver support system would ideally assume the functions of the liver and prevent the clinical manifestations of the disease.

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There are two main categories of disease in which this may be useful. The first is to treat reversible liver disease, in which ECLS would allow time for recovery and regeneration. The second is in the management of irreversible liver disease in which it may be used as a bridge to transplantation or to control symptoms, such as puritis, in patients not suitable for transplant.

In ALF, the prevention of multi-organ failure to allow time for the liver to regenerate may mean that liver transplantation is avoided. An optimised physiological state may actually promote liver regeneration. Alternatively if recovery does not occur, the progression towards fulminant hepatic failure may be slowedand physiological optimisation is desirable whilst awaiting transplantation. In ACLF the ECLS may bridge the decompensated period, promoting liver regeneration.. Similarly, in SFSS the period of inadequate activity may be managed with ECLS allowing time for regeneration of the liver in the live-donor, the post-operative patient and the transplant recipient. In CLF the ECLS may serve to ameliorate symptoms such as intractable pruritus. Scientific basis for ECLS

An extra-corporeal system is one that removes blood from the body, performs the desired function outside of the body and returns purified blood back to the circulation. Most organ support strategies aim to replace a sufficient amount of organ function during a period of organ failure to optimise the chances of recovery, prevent multi-organ failure or death. This process is routine in the management of renal failure to detoxify the blood of urea, creatinine, electrolytes and other small to medium sized water soluble molecules. Another example includes the management of gas-exchange or ventilator failure by removing carbon dioxide and the supplying oxygen with an extracorporeal membrane oxygenator. The difficulty with replacing ‘liver function’ is that this encompasses a vast number of independent functions. ECLS must detoxify, synthesise protein, perform metabolic functions, regulate immune activity and remove cytokines to prevent a pro-inflammatory state. As the regenerative ability of the liver is well appreciated, initial ECLS systems were aimed at providing short-term support to allow this to happen. Producing an ECLS that can provide biosynthesis is important but it is not crucial. It is possible to replace many of the substances that are formed and regulated by the liver by the administration of blood products and medications. For example glucose, albumin and coagulation product therapy may support a liver unable to synthesise these substances. Therefore, the more challenging aspect of ECLS is detoxification. The two early approaches in the 1970s were based around these principles. These included filtration of blood for detoxification in combination with exchange transfusions and extra-corporeal cross-circulation with either human or animal hepatocytes. This led to the development of two categories of ECLS. The first category is an artificial filter aimed at detoxification. The second is a cell-based biological device, which aims to replace synthetic function.

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Artificial Devices

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Extra-corporeal detoxification devices are already widely available as RRT. Dialysis or haemofiltration are effective at removing water-soluble small and medium sized molecules from the circulation. These devices work by allowing water-soluble un-bound substances to pass across a semi-permeable membrane into hypotonic dialysate fluid along their concentration gradient (26) (Figure 1a). However many of the substances that accumulate in liver failure are protein bound or have a high molecular weight (MWt) and are therefore not readily removed by conventional dialysis or filtration techniques. The difficulty with removing molecules bound to albumin is that circulating albumin has a much higher affinity for these molecules than the dialyslate solution on the other side of the semi-permeable membrane. In addition, the semi-permeable membrane itself has no ability to aid dissociation of the toxin from albumin and thus only the small amount of unbound toxin is removed. As such removal by ECLS devices depends of the dissociation kinetics of the molecule from albumin. [FIGURE 1A]

Plasmapheresis (plasma-exchange) A simple approach to albumin cleansing is simply to remove and replace it. Plasmapheresis is an established treatment modality for various autoimmune conditions and is gaining evidence in the treatment of ALF. Plasma is separated from cells in an extra-corporeal device, which uses either a filter or centrifuge. The plasma is then replaced with either human albumin solution (HAS), fresh frozen plasma (FFP) or a combination of the two. It effectively removes albumin and albumin-bound toxins, cytokines and antibodies. High-volume plasma exchange (> 10 L /day) has demonstrated an improvement in hepatic encephalopathy grade (HE) and mortality in patients with ALF (27). A new system currently being developed is incorporating low-volume plasma-exchange with albumin dialysis Plasma Filtration

An alternative to plasma exchange, is to filter the plasma using a plasma filter, but rather than discarding the filtered plasma, but to pass the filtered albumin through an adsorption column(s), typically carbon or resin, designed to absorb endotoxins, small proteins and other compounds, including nitric oxide, and then return the plasma to the patient. Haemoperfusion: Charcoal-dialysis and Albumin-Dialysis

Haemoperfusion may be described as the process of passing blood directly over an absorbent substance to remove toxins. In contrast, haemodialysis uses the property of diffusion to clear molecules. The first generation of haemoperfusion

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devices used charcoal as the absorbent material. However these devices often caused profound thrombocytopenia and recurrent circuit clotting due to the bioincompatibility of the charcoal. In addition these early charcoal perfusion caused hypersensitivity reactions and charcoal embolization, and were subsequently withdrawn. An alternative approach was to add the charcoal powder into the dialysate solution, so that there was no direct blood contact (28). This technique was able to attract large MWt molecules bound to albumin and cytokines to pass across the semi-permeable membrane and irreversibly bind them via hydrogen bonds and Van Der Waals forces to the charcoal dialysate. Although animal studies demonstrated biological efficacy, human studies failed to demonstrate a significant clinical benefit (28).

Albumin replaced charcoal as the medium for haemoperfusion systems (plasmaperfusion or albumin dialysis). However it was still only effective in removing non-albumin bound toxins from the blood stream. Consequently, this system did not alter clinical outcome measures and was also associated with complications including hypersensitivity reactions and coagulopathy (29).

The failure of albumin dialysis was due to the inability to rapidly separate bound toxins from circulating albumin. In part this was due to the use of simple semipermeable membrane haemofilters that had no intrinsic absorbency to encourage this separation. Thus subsequent efforts were directed to combine the safety of the haemofilter (keeping blood and dialysate separate) with the biological activity of the haemoperfuser (blood perfused over an absorbent substance). This was achieved by separating the dialysate from the blood with an intermediate filter which itself could encourage movement of albumin bound solutes.

This led to the development of newer designs for haemofilter membranes, with the creation of a lipophilic membrane that consisted of a polysulfone filter filled with paraffin (30). The idea behind this design is that lipid-soluble molecules will be forced to dissociate from albumin and preferentially bind to this filter. However using this paraffin containing haemofilter did not work in practice, but led to the development of an albumin-impregnated polysulfone high-flux filter (31). This allowed albumin-bound toxins to dissociate and bind to the albuminimpregnated filter. Albumin dialysate on the other side of the filter would then increase removal of these toxins, so regenerating the albumin-impregnated filter, thus allowing continuing activity. Experimental studies demonstrated efficacy using this technique and was originally used early systems (see below). However more recently developed, high-flux membranes, have been shown to be more effective at detoxification than albumin-impregnated filters. The albumin dialysate may either be used once and discarded or regenerated by being passed through an absorbent material. Single Pass Albumin Dialysis (SPAD)

SPAD represents the simplest form of albumin dialysis and may be used with conventional haemofilters. The patient’s blood passes through a high flux filter, impermeable to albumin (used in RRT) and is dialysed against an albumin

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solution to remove small, protein bound molecules. To increase efficiency an albumin-impregnated filter may be used. The albumin dialysate is discarded once used (Figure 1b,1c). [FIGURE 1B] [FIGURE 1C]

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Molecular absorbent re-circulatory system (MARS) MARSTM (Gambro AB, Lund, Sweden) utilises albumin dialysis through an albumin-impregnated semi-permeable membrane combined with haemofiltration and regeneration of the albumin dialysate. The membrane allows molecules of less than 50 KDa to cross, thus preventing albumin loss. Lipophillic protein-bound substances first bind to the membrane and then are subsequently removed by a 20% human albumin solution (HAS) containing dialysate. The dialysate is then circulated through a charcoal haemofilter and anion exchanger to regenerate the albumin and a haemodiafiltration system to remove smaller, water-soluble molecules. Thus the absorbent charcoal is separated from the patient’s blood, improving the safety profile (Figure 2). However as with any adsorption system MARSTM loses detoxifying ability after about 6 hours of use due to exhaustion of sorbent binding. [FIGURE 2]

Fractionated plasma separation and absorption (Prometheus) and select plasma exchange therapy (SPECT). Albumin may be detoxified in-vivo (whilst remaining within the circulation as with the MARSTM system) or in-vitro (by temporarily separating albumin from the blood). Separating the plasma from the circulation allows detoxification in a separate extra-corporeal circuit and then returning it to the circulation is the basis behind the PrometheusTM device (Fresenius Medical Care, Bad Homberg, Germany).

To achieve this, a large pore filter is required with a pore size of 250-300 KDa. This allows albumin to pass through into an extra-corporeal circuit. The plasma filtrate is then directed through a neutral resin absorber and an anion exchanger for detoxification before returning via a haemodiafilter to the systemic circulation (Figure 3). PrometheusTM also retains ability to detoxify for around 6 hours before the sorbents become fully saturated. The plasma filtrate passes through two absorbers, and these provide unselected removal of plasma proteins. So although they may remove toxins, they also remove some normal plasm proteins, such as protein C. As protein C levels are reduced in patients with ALF and ACLF (32), and even lower in those patients who develop acute kidney injury (33), so the risk of clotting in the extracorporeal circuit is increased (34).

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Alternatively the plasma can be discarded and replaced with HAS. This is the basis behind the select plasma exchange therapy (SPECT) device which again uses a hollow-fibre membrane with a pore size of 100 KDa to allow albumin passage into the extra-corporeal circuit whilst retaining coagulation factors, immunoglobulins and stimulators of hepatic regeneration.

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Clinical Outcome studies

The majority of the clinical studies investigating artificial liver support devices have been performed in patients with ACLF with much studies reporting on patients with ALF. Most of these studies are limited as the number of patients included are small and are predominantly case-series or retrospective analyses (table1). Three large randomised controlled trials (RCTs) have recently been published (FULMAR, HELIOS and RELIEF) which are discussed below. Physiological efficacy

Reviewing clinical trials of ECLS devices then the MARSTM albumin dialysis has been the most studied. Both the MARSTM and the PrometheusTM systems have been shown to clear protein-bound toxins, bilirubin, urea and creatinine in patients with ALF and ACLF (35,36). In addition, circulating neurohormones, and nitric oxide are also cleared, and both free radical production and markers of oxidative stress are decreased. Even though ammonia levels remain unchanged, hepatic encephalopathy grades improve (36). There have been few clinical studies directly comparing MARSTM and PrometheusTM devices, and as expected from the extracorporeal circuit design PrometheusTM generally provides greater clearance of toxins, bilirubin, urea and creatinine then MARSTM (37). PrometheusTM will also remove unconjugated bilirubin, which is not removed by MARSTM (38). Both PrometheusTM and MARSTM require a secondary extracorporeal circuit, to eliminate small water soluble solutes, whereas this is a haemodialysis circuit with PrometheusTM, it is typically a haemofiltration circuit with MARSTM, so small solute clearances, such as urea and creatinine would be expected to have greater clearances with haemodialysis. Patients with ALF and ACLF are at risk of hypoglycaemia, and as such dialysates should contain glucose to prevent hypoglycaemia. Hepatic Encephalopathy & Intracranial Pressure

Treatment with intermittent haemodialysis and haemofiltration has been shown to cause an increase in intracranial pressure (ICP) in patients with ALF (39), although ICP is more stable with continuous forms of renal replacement therapy (40). Animal studies have been published demonstrating a decrease in intracranial pressure (ICP) in pigs with ALF receiving intermittent MARSTM and PrometheusTM support (41,42). A small study in patients with ALF reported that ICP did not increase with intermittent MARSTM treatments, and this was not due to thermal cooling (43). Several other studies have reported improvements in

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the grade of clinical hepatic encephalopathy (HE) using MARSTM in patients with ACLF (36, 44, 45). However despite the stability of ICP and reduction in HE only one study has demonstrated a reduction in HE coupled with improved patient survival (45).

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Hepato–Renal Syndrome

HRS is typically described as a vasomotor nephropathy, although HRS is associated with inflammatory changes within the kidney (46). Both MARSTM and PrometheusTM have been used to treat patients with ACLF and HRS type 1. Small case series reported an improvement in renal function and creatinine clearance (47, 48). In a secondary post hoc subgroup analysis of patients with HRS recruited into the HELIOS study, a survival advantage was demonstrated for those patients receiving PrometheusTM support (49). Safety and biochemical efficacy was further demonstrated in a recent a case series in which patients with HRS were treated with Prometheus (50). To investigate this further there is a current ongoing prospective trial (LUTHOR study universal trial number U1111-1115-4645). Cholestatic Pruritus

Improvement in cholestatic pruritus has been described in case reports using both MARSTM and PrometheusTM treatments(51), and in the UK MARSTM treatments have been approved by the National Institute of Clinical Excellence (NICE).for treating cholestatic pruritus refractory to standard medical therapy. Patient Survival

Clinical improvement and greater short-term survival have been reported for MARSTM treatments in small series of patients. However a prospective case– controlled study of 13 patients receiving intermittent MARSTM treatments observed no improvement in survival, even though toxins were removed efficiently and hemodynamic parameters improved significantly (52). A metaanalysis similarly was unable to demonstrate any significant survival advantage using MARSTM treatments in liver failure compared with standard medical therapy (53).

The FULMAR study, a randomized controlled multicenter trial in 16 French centers evaluated the efficacy and safety of MARSTM in 102 patients with fulminant and sub-fulminant hepatic failure. There was a trend towards improved 6-month survival in the MARSTM treated group although this did not reach statistical significance. The interpretation of the results is somewhat challenging however as 69% of patients were transplanted very quickly (mean 16 hours), leaving only a very short period in which to assess the response to the intervention (54). In the RELIEF study, 189 patients with ACLF were randomized to receive standard treatment or support with MARSTM. The MARSTM cohort had more patients with higher model of end-stage liver disease (MELD) scores and

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spontaneous bacterial peritonitis, which were two factors found to be independent risk-factors for mortality. No survival benefit could be demonstrated despite physiological improvement (55). A similar prospective trial was performed with the PrometheusTM device in the HELIOS study. Again no overall survival benefit was observed compared to standard medical therapy, but post hoc secondary analysis showed that in the sub-groups of patients with HRS and high MELD scores, there was a marginally significantly statistical improvement in 28 day survival (49).

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The safety of both these ECLS devices is well established with more than 5000 patients treated., Commonly reported side effects including thrombocytopenia, leucocytosis, bleeding, hypotension and filter clotting.

Other ECLS devices have been investigated less extensively. Single pass albumin (SPAD) has not been prospectively studied in any multicentre trial. Case-reports and small case series report an improvement in biochemical parameters (56). Similarly SPECT has been reported to produce promising survival outcomes in animal models, but as yet there have been no human RCTs (57).

Plasma exchange has been shown to reduce the levels of circulating cytokines, and inflammatory mediators and products from necrotic cells (DAMPS). Clinically plasma exchange has been observed to reduce the degree of HE and improve haemodynamic and ICP stability in ALF. Recently Larson and colleagues published findings suggesting that daily high volume plasma exchange with fresh frozen plasma replacement improves survival in patients with ALF, particularly for those not receiving liver transplantation (27). Whether this is due to removal of inflammatory mediators or replacement of plasma proteins remains to be determined. Biological-artificial (bioartificial) Devices

Arguably a more elegant solution would be to replace all functions of the failing liver to a sufficient degree to allow normal physiology to be maintained. Bioartificial devices incorporate two components. The biological component is a hepatocyte or another cell-line capable of performing the same biological functions. Ideally cells would remain terminally differentiated to maintain hepatocyte-specific function and remain stable in-vitro. The artificial component is a hollow-fibre membrane, similar to the devices above, which allow exchange from plasma between the patient and the matrix containing the hepatocytes. These two components form the bioreactor. The mass of hepatocytes needed is uncertain, but based on studies on patients undergoing hepatic resection approximately 150-400g of hepatocytes, or 1010 cells, are required to achieve 10-30% of the functional capacity of the normal liver. The ideal biological component would obviously be human hepatocytes, however commercial cell culture is difficult, supply is limited and hepatocytespecific function deteriorates rapidly with repeated passages. Porcine hepatocytes are incorporated into some devices (see below) but are less stable and risk transfer of cross-species disease, in particular porcine retroviruses. An

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alternative approach is to use cell lines that have been genetically modified to achieve functional and survival criteria. The C3A cell line derived from hepatoblastomas are easily cultured and readily available, but lose hepatocytespecificity quickly, are unable to perform complete ureagenesis and potentially risk malignant contamination. An immortalised human hepatocyte cell line, HHY41, retains more function and is currently being investigated.

The bioreactor design in its simplest form is a hollow-fibre capillary though which the patient’s blood or plasma flows, surrounded by an extra-capillary matrix containing the hepatocytes. To increase efficiency of transfer, plasma is often separated from blood in a separate circuit with a large pore haemofilter (50 – 150 KDa) before entering the bioreactor. The addition of albumin-dialysis and haemodiafiltration to assist with detoxification is also possible. A separate capillary to supply oxygen to the hepatocyte matrix is also required. Different arrangements of hepatocytes within the bioreactor exist including tissue slices, homogenates, spherocytes or in suspension.

The bio-artificial liver support system (BLISS) and the Academic Medical Centre Bio-Artificial Liver (AMC-BAL) use porcine hepatocytes. The Hepat-assist device uses a similar approach but employs two charcoal columns for detoxification prior to a bioreactor containing porcine hepatocytes. The Modular extracorporeal liver system (MELS) passes fractionated plasma through human hepatocytes. The ELAD (Vital therapies, USA) uses C3A human hepatoblastoma cells in suspension around hollow-fibre capillaries through which the patient’s separated plasma passes. Clinical outcome studies

Most trials involving BALs have been performed in patients with ALF. The three devices that use porcine hepatocytes as the biological component are the AMCBAL, the Hepat-assist and the BLISS.

The AMC-BAL was first developed in 1996 (58) and was subsequently used in 12 patients with ALF and 8 patients with ACLF (59). The patients with ALF were all successfully bridged to liver transplantation whereas only 2 of the patients with ACLF were successfully bridged. A subsequent case series identified neurological improvement in 60% of cases with a reduction in serum bilirubin and ammonia, however there were significant complications related to bleeding and haemodynamic instability (60). However a multi-centred randomised control trial concluded that there was no survival advantage in fulminant or subfulminant liver failure. Whilst there was some improvement in neurological status, there was little evidence of improved synthetic function (61). The Hepatassist has been assessed in a small series of patients with ALF and demonstrated improved survival at 30 days (62), but has not been tested in a larger multicentre prospective trial.. The ELAD, using hepatoblastoma cells, has been studied in a number of human trials (63, 64). Overall, plasma ammonia and bilirubin concentrations were reduced in patients being managed with ELAD and in a subsequent,

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underpowered trial there was a survival advantage demonstrated in patients with ACLF who subsequently underwent liver transplantation (64). There have been a number of international trials of the ELAD device predominantly in patients with ACLF, and acute alcoholic hepatitis which are expected to report shortly. The company’s phase 3 trial results (ELAD VTI-208) have just been announced. In a randomised, open-label trial 203 patients with alcohol induced liver disease were treated either with ELAD (96 patients) or conventional therapy (107 patients). There was no difference in 90-day survival, but a posthoc analysis identified that patients under the age of 50 years old and with normal renal function may benefit (65). A further trial to investigate this subgroup is planned. The MELS device, using human hepatocytes, was studied in patients fulfilling criteria for liver transplantation, in which all patients were successfully bridged to transplant (66). However there have been no recent multicentre trials with this device.

On the positive side there have been no reports of transfer of porcine retroviruses using porcine hepatocytes or transfer of hepatoblastoma cells causing malignancy. On the other hand anticoagulation of these bioreactor ECLS, particularly for those systems in which there is direct blood contact have at times proved problematical, with bot excess circuit clotting and bleeding, especially when heparin has been used as the anticoagulant. Timing of intervention

The decision to start liver assist therapy may be crucial in the modulation of mortality. It has been well established that MARS™ and other devices have beneficial effects on physiology and organ systems, preventing the progression of multi-organ failure. These improvements have not necessarily been translated into survival advantages for patients with ACLF. Comparing two small trials involving the MARS™ device in which the initiation of treatment differs, highlights the importance of timing. In the first study (45) the mortality in the control group was 45% compared to the mortality in the MARS™ group of 10%. In a second study (67) control group mortality was 100% and MARS™ group mortality 80%. One interpretation would be that introducing ECLS support early before established organ dysfunction has occurred to prevent the progression of multi-organ failure is more likely to allow these devices to influence clinically relevant outcomes. However this assumption requires testing prospectively. New Technologies

Disappointingly the physiological efficacy of these ECLS devices have not necessarily been translated into increased patient that many clinicians had hoped for. The reasons for this are likely to be complex and in-part due to our incomplete understanding of the pathophysiology that results in multi-organ failure and eventually death. As understanding of this complicated disease process evolves it is likely that these devices will play a greater role in the management of patients with liver disease. After the initial disappointing trials of

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biological devices, it was assumed that failure was due to an inability to produce bioreactors containing an adequate number of biologically functioning hepatocytes. The current generation of bioreactors now have hepatocyte mass equivalent to around 30% of normal liver. As technology advances it is likely the biological devices will maintain their cell activity and differentiation for longer periods. However it is likely that hepatocytes grown on bioreactors do not function in an identical fashion to native hepatocytes. We now realise that not only does the liver contain other cell types, endothelial cells, interstitial cells of Ito, and Kuppfer cells, but also has two different blood supplies, hepatic artery and portal vein. Advances in the understanding of hepatocyte biology has demonstrated that hepatocytes have different functions depending upon their position within the liver; peri-arteriole, peri-venule and peri-bile duct. As such the next generation of biologic devices may have to develop constructs mimicking the structure of the hepatic lobule to induce cell-cell interactions. Recent trials of new spheroid bioartificial biological devices in animal models have reported promising results (68). Improvements in manufacturing processes will hopefully reduce costs and increase availability so allowing earlier initiation of therapy to preventing the cascade of organ failure that becomes increasingly difficult to reverse. Just as there is continuing research into developing bioreactors, there have been advances in extracorporeal circuits, with promising reports of combinations of low volume plasma exchange and plasma filtration with absorption in large animal models (69). Other researchers have combined a high permeability haemofilter with an endotoxin absorption membrane dialyzer (70). Obviously these devices will require testing in appropriately designed prospective clinical trials (71).

Expert commentary The authors have personal experience of using both simple extracorporeal circuits; plasma exchange, continuous renal replacement therapies, single pass albumin and MARS™ dialysis, more complex extracorporeal circuits combing high permeability dialyzers with absorption membranes, and bio-artificial devices [71]. One of the key problems is that patients with ACLF and ALF are prothrombotic [32,33], and as such maintaining patency of extracorporeal circuits is often more difficult than for the general ICU patient [34]. Some circuits, particularly those which use a superflux highly permeable dialyzer in combination with anionic absorption columns [72], remove protein C so increasing the risk of clotting within the circuit. As most patients have reduced levels of anti-thrombin, then anticoagulation with heparin is not as effective, and as such other anticoagulants including citrate and prostanoids are often required [73,74]. Although citrate is metabolised by the liver , and as such citrate toxicity may more frequently occur in patients with ALF, particularly with acetaminophen toxicity, citrate anticoagulation is possible in many cases of ACLF, and the risk of citrate toxicity can be reduced by using continuous renal replacement circuits in dialysis mode, increasing dialysate flow rate and reducing blood flow, to maintain a ratio of total calcium to ionised calcium of less than 2:1.

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5 year view There has been renewed interest in high volume plasma exchange [75], and there will be a series of trials designed to determine whether the encouraging results reported for treating patients with ALF, are due to removal of toxins and inflammatory mediators, or the replacement with fresh frozen plasma, and identifying key anti-inflammatory mediators in pooled plasma. The debate on the role of albumin continues [76], with the development of new devices now becoming available for clinical use. In liver failure albumin fails to act as a detoxifying agent, as binding sites become full, and albumin changes shape, so simply trying to remove bound toxins, does not refresh albumin. We expect further developments to improve the binding capacity for toxins of both exogenously administered albumin, by removing stabilising agents which had been added to allow for longer shelf storage of commercially available albumin solutions, and also research into reconfiguration of endogenous albumin. Although there have been positive results with some of the bio-artificial devices [77,78], the majority of recent trials have not been positive. However there have been marked improvements in the designs of bio-artificial constructs, and whereas the traditional biological device has comprised a single cell type, such as hepatocytes, these newer constructs allow cell-cell interactions between different cell types, so allowing more complex devices to be developed. At the moment these are been trialled in animal models, for example a bio-artificial spleen, which can remove bacteria form plasma [79]. It is therefore most likely that with the next 5 years, more complex bio-artificial livers will be developed, that not only reproduce some of the differences in hepatocyte function with the hepatic sinusoid, but also include bile ducts and macrophages, so working towards a liver as an organ, rather than a simple array of hepatocytes. Until these devices are developed we expect that more complex extracorporeal circuits, combing a highly permeable dialyzer with endotoxin absorption, and then incorporating re-infusion of albumin and fresh plasma protein will become the dominant extracorporeal support for patients with ALF and ACLF. Tangential developments in reducing the risk of clotting in the extracorporeal circuit, by modulating the surfaces of venous catheters and blood lines will improve the effectiveness of circuits, by extending treatment times [80]. Conclusion Liver disease is an increasing public health concern. The liver is a complex organ with a myriad of functions and interactions throughout the body, which results in a challenging organ to emulate with an extra-corporeal device. Early devices focused on haemoperfusion to simply remove accumulating toxins. This evolved into albumin dialysis and subsequently into devices designed to perform the metabolic and synthetic functions of the liver as well. Despite promising physiological effects, improved patient survival has as yet not been convincingly demonstrated. The reasons for this are likely to be multi-factorial but may be related to our understanding of disease, our technological ability to reproduce organ function and the timing of our therapy, limited by logistic reasons. Ideally these devices could bridge patients to liver transplant, support a patient whilst

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spontaneous recovery occurs or provide symptomatic relief in patients with untreatable, end-stage disease.

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Key Issues 1. The mortality from liver disease has increased over 4 times in the last 50 years. The spectrum of disease includes acute liver failure, acute-on chronic liver failure and chronic liver failure. The causes for these conditions are diverse. 2. Due to the complexity of the functions of the liver, efforts to produce an extra-corporeal support device have been less successful than for other support devices (such as renal replacement therapy or extra-corporeal membrane oxygenation). Such a device may be useful to allow a liver to regenerate, providing bridging therapy for patients awaiting liver transplant or ameliorate the symptoms of liver failure as a stand-alone therapy. 3. The main challenge is to produce an effective system for detoxification. Two extra-corporeal approaches exist. The first is an artificial filter which allows movement of albumin-bound toxins across a semi-permeable membrane. The second is a biological approach utilising human or animal hepatocytes to simultaneously detoxify and biosynthesis. 4. Artificial devices, such as MARS and PrometheusTM, use an extracorporeal membrane which allows albumin-bound toxins to pass through to a dialysate. This dialysate is human albumin solution which becomes saturated with the filtered toxins. To maintain efficacy the albumin solution needs to be regenerated with ion exchangers and carbon columns, or replaced with a fresh supply of albumin. 5. These devices have been extensively investigated, which are discussed in detail. Biochemical efficacy and detoxification has been repeatedly demonstrated. In addition a reduction in intra-cranial pressure and a possible benefit in the management of hepato-renal syndrome has also been shown. No mortality advantage has been shown with the use fo these devices. 6. Biological devices (or bioartificial devices) use hepatocyte cells from human or animal sources. These devices combine a filter with a column, which contain 150-400g of cells. 7. The bio-artificial devices have also demonstrated efficacy in detoxification and improvement in neurological status, but have not consistently impacted on mortality. A recent Phase III randomised trial has failed to demonstrate improved survival at 90 days. 8. Future work may concentrate on particular sub-groups of patients who may benefit from extra-corporeal liver support. In addition the timing of therapy may be vital with therapy commenced before the progression of multi-organ dysfunction likely to be most successful. The development of new filters and a more robust source of hepatocytes may improve the outcome for patients requiring support therapy.

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Financial and competing interests disclosure

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The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Table 1A. Artificial Devices Device

Principles of therapy

Clinical Studies

SPAD (Single-Pass

Albumin dialysis against

Improvement in biochemical

Albumin Dialysis) MARS (Molecular

Adsorbent Recirculating System)

2-5% albumin

Albumin dialysis against 20% albumin.

parameters, comparable

with MARS. Only single case studies available,55 no RCTs Improved hepatic

encephalopathy36, improved quality of life, no significant survival benefit.9

28 Prometheus (Fractionated Plasma separation,

Improvement in biochemical

Adsorption - Prometheus) resin and anion

benefit at 28 days.69

Plasma Separation and

SEPET (Selective Plasma Filtration Technology)

adsorption using neutral adsorbers

100 kDa hollow fiber

No human RCTs. Animal

fresh-frozen plasma

survival. 70

membrane, albumin and mixture as replacement

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parameters. No significant

models show improved

fluid HVPE (High Volume Plasma Exchange)

Patient’s plasma

removed and replaced

with fresh frozen plasma

Improved transplant-free survival in ALF.27

29

Table 1B. Bio artificial Devices Device

Principle & Cell

Main concern

Clinical Studies

Zoonoses

176 patients, no

Type Hepat Assist

Plasma separation,

charcoal adsorption,

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porcine hepatocytes

fulminant and sub-

MELS (Modular

Plasma separation

Supplies low,

Liver System)

through human

to maintain

Extracorporeal

then plasma passed hepatocytes

ELAD

Human hepato-

Liver Assist

cells)

(Extracorporeal Device)

BLSS (Bio

artificial Liver

blastoma cell (C3A

Porcine hepatocytes

fulminant61

8 patients, successfully

function difficult bridged to transplant57

Tumorogenicity

6 human studies, 150 patients treated.

Survival benefit in

ACLF study in 49 pts.71 Zoonoses

Support System) AMC-BAL

survival advantage in

Phase I study in 4

patients, no serious adverse events.72

Porcine hepatocytes

Zoonoses

12 patients treated, 11

bridged to transplant.51

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30

Fig1a

Fig1b

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31

Fig1c

Fig2

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32

Fig3