Hepatobiliary Quiz-3 (2012)

Answers to Multiple Choice Questions

1. Correct answers: 3 and 4

Based on >8% structural variability in the whole genome sequence, HBV can be divided into eight different genotypes (A–H) which are distributed in different geographical regions of the world.¹ Recently, two new genotypes have also been reported: genotypes I and J. Genotype I is a recombinant between genotypes A, C, and G which has been isolated from Laos and Vietnam.² Genotype J has been reported from the Ryukyu islands, Japan.³ Because of the geographical sequestration of various genotypes, direct comparison of the clinical features between all the genotypes is limited. However, there is information regarding the clinical behaviour of genotype B vs C and genotype A vs D, since these genotypes are clustered together in a geographical area. Genotype B is associated with milder disease, is more likely to be HBeAg negative and have less risk of development of hepatocellular carcinoma compared to genotype C.^4–6 The studies comparing the clinical outcomes and clinical severity of the disease between genotypes A and D have presented conflicting results without any clear result on which genotype is associated with more severe disease.¹ There is a gradation of e-antigen clearance/e-antigen seroconversion response when treated with pegylated interferon (IFN) according to the genotypes: SVR was 44.3%, 31.5%, 29.1%, and 24.4% among genotypes A–D-infected patients, respectively. In addition, long-term HBsAg loss/seroconversion was also significantly higher among genotype A infected chronic hepatitis B patients [both e-antigen-positive (15.3%, 1.7%, 2.2%, and 1.6% for genotypes A–D, respectively) and e-antigen-negative (20%, 6%, 9%, and 6% for genotypes A–D, respectively)] when treated with pegylated IFNs.^7–10 However, a meta-analysis has shown that the response to oral nucleos(t)ides is not dependant on genotype.¹¹

2. Correct answers: 1, 4 and 5

In liver transplantation, a liver mass amounting to approximately 1% of the weight of the recipient is required to be implanted for maintaining basic metabolic functions of the liver in the postoperative period. This is usually expressed as a graft recipient weight ratio (GRWR) of 1%. Small-for-size syndrome refers to the constellation of persistent ascites, cholestasis, coagulopathy, and encephalopathy in the setting of a partial liver graft with GRWR < 0.8% without a technical cause. Small for size syndrome (SFSS) results in higher incidence of septic complications and increased mortality. By and large, it is agreed that SFSS can be avoided if GRWR is greater than 1%, and there is an increased frequency of SFSS when GRWR is <0.8%.^12–14 A left lateral segment of a 60 kg adult typically weighs around 250 g (20% of the total liver weight) and would suffice for children weighing up to 25 kg; thus SFSS which is more common when left lobe grafts are used in larger adolescents and adults.¹⁵ There is human and animal evidence implicating portal hyperperfusion in the pathogenesis of graft injury in SFSS. The partial grafts in living donor liver transplantation (LDLT) are subjected to the portal flow destined for a whole liver, resulting in portal hyperperfusion. Recipient haemodynamics too plays a part, as cirrhotic recipients demonstrate higher portal flow due to splanchnic vasodilatation. This leads to microvascular injuries to the graft.^16,17 Hence, most clinical strategies to avoid SFSS have focused on ways to decrease portal hyperperfusion in the new graft, including shunts to decrease portal blood flow, splenic artery ligation and splenectomy.¹⁵ Splenectomy is probably the most widely practiced modality now for portal flow modulation as it is technically familiar to most surgeons.^18,19

Despite the inherent disadvantage of left lobe grafts in predisposing to SFSS, they do have certain advantages which make them attractive options in LDLT. Left lobe grafts have better venous outflow drainage from the middle and left hepatic veins which drain via a common orifice which can be simply implanted into the recipient inferior vena cava. Almost all left lobe grafts have single biliary hepatic duct orifice compared to multiple orifices in right lobe, allowing for easier biliary anastomoses and less biliary leaks. Similarly, left portal vein is usually single and longer ensuring an easier anastomosis in the recipient. The morbidity to the donor after a left lobe resection is less than after right lobe resection.¹⁵

3. Correct answers: 1 and 2

Non-alcoholic fatty liver disease (NAFLD) is ubiquitous disease affecting all parts of the globe with a prevalence of about 10–30% in most societies. Whether residing in the East or the West, Asians seem to be more at risk than their Caucasian and African American counterparts.²⁰ In a multiethnic study from the USA, Hispanics had disproportionately higher representation in the NAFLD group compared with base population and whites and African Americans had lower representation, suggesting that Hispanics had the highest risk of developing NAFLD, followed by Asians and least in Whites and African Americans.²¹ While Caucasian patients are more likely to be older, female, and obese, Asians develop NAFLD at a younger age, are more likely to be male, and are less likely to be obese. Within the USA, Asians have a lower age at presentations (46 years) compared with Caucasians (52 years) and African Americans (61 years).²¹ Among Asians, 79% of NAFLD patients are male, compared with only 44% among Caucasians.²¹ Mean BMI in Western studies of patients with NAFLD has been around 30–35 kg/m². This is in sharp contrast to Asian studies where the mean BMI is about 29 kg/m² in India; 24 in Korea, 27 in Sri Lanka, 23 in China, and 23 in Japan.²⁰ Traditional risk factors for NAFLD including advanced age, obesity, diabetes, and MS are less common in Asians. Thus, there is a strong empirical reason to suspect additional risk factors such as a genetic predisposition for the development of NAFLD in Asians. A study from the USA found that in healthy non-obese subjects, 38% of the Asian Indian men with apolipoprotein (APO) C3 promoter region polymorphisms (SNPs) (C-482T and T-455C) had NAFLD and marked insulin resistance (IR), whereas no APO C3 wild-type homozygotes had NAFLD. In the non-Asian Indian cohort, the difference in NAFLD between those with and without variant alleles was 9% and 0%, respectively.²² In contrast, other studies in various ethnic groups like Americans, European Americans, African Americans, and Hispanics have shown that APO C3 variants probably have no effect in causing NAFLD, nor are associated with IR.^23,24 Even other Asian ethnicities like Chinese did not have this association.²⁵ This discrepancy could be due to racial differences which make Indians more susceptible to the effects of APO C3 polymorphisms. Patatin-like phospholipase domain-containing protein 3 (PNPLA3) SNPs seem to be strongly associated with NAFLD irrespective of ethnicity. A recent meta-analysis has confirmed not only the role of PNPLA3 polymorphism in the development of NAFLD across all ethnicities, but also its association with more severe disease with 3.24 times higher risk of high necro-inflammatory scores and liver fibrosis in GG compared with CC genotype.²⁶

4. Correct answers: 1, 3 and 5

Hepatocyte transplantation is being developed as an alternative treatment for the management of acute liver failure (ALF) and liver-based metabolic disorders. It has several advantages: (a) less invasive, with less morbidity and mortality, (b) the native liver remains in situ allowing the possibility of regeneration over time and recovery in the setting of ALF, or at least buying the patient time until a suitable organ is available for transplantation, (c) can be performed multiple times, (d) cryopreserved hepatocytes can be used urgently and (e) one donor can benefit multiple recipients.²⁷ However, similar immunosuppression like liver transplantation is required. Before transplantation, the hepatocytes must fulfil certain criteria: (a) blood group ABO compatible, (b) have cell viability of ≥60%, (c) negative viral screening as for whole organ transplantation, and (d) micro-organism free on Gram stain immediately prior to administration.²⁷ Cell viability is checked for by tryptan blue test in which the dead cells take up the blue stain. Up to 2.4 × 10⁶ cells/g native liver may be transplanted, with a maximum total number of hepatocytes of 5 × 10⁸ per infusion. The goal is to transplant a total mass of cells equivalent to 10% of the recipient's liver mass or 1 × 10⁸ hepatocytes/kg body weight.²⁷ It is best to transplant hepatocytes fresh, but if no patient is waiting, these cells can be cryopreserved using optimized protocols, though there will be some loss of metabolic function on thawing.²⁸ There are several possible sites suitable for cell transplantation; however, the preferred route is intraportal. Portal circulation can be accessed by the direct portal vein puncture, the operative insertion of a central venous catheter in portal venous tributaries, or the umbilical vein in neonates. The portal pressure should be measured after every infusion (infusion time 5–10 min), and subsequent infusions to continue as long as the portal pressure stays below a persistent increase of 12 mmHg. The splenic vein can be used as the spleen is an important extra-hepatic site for engraftment of hepatocytes and with time bile canaliculi, sinusoids and endothelial cells develop in spleen. This is useful in cirrhotics or acute liver failure with ongoing massive necrosis where the liver is not a hospitable environment for transplanted cells.²⁹

5. Correct answers: 2, 4 and 5

Pre- treatment predictors of favourable response to treatment of chronic hepatitis C (CHC) include genotype non-1 infection (mostly genotypes 2 and 3), viral load of <600,000 IU/ml, female gender, age <40 years, non-African-American race, lower body weight (≤75 kg), absence of insulin resistance, elevated alanine transaminase (ALT) levels, and absence of bridging fibrosis or cirrhosis on liver biopsy.^30,31 IL28B polymorphisms were identified on the basis of genome-wide association studies to be predictors of response to treatment in CHC in various ethnicities, including Caucasians, Asians, African Americans and Hispanics.^32–35 Most of these studies are in genotype 1 patients and the role of these polymorphisms in genotype 2 and 3 is still controversial.³⁶ Further, it has also been shown that favourable genotypes have higher rates of spontaneous clearance of hepatitis C after an acute infection.^37–39 The IL28B gene encodes IFN-λ3, which is involved in the early host innate immune response to HCV, and may explain the observed effect on spontaneous clearance.³⁶

6. Correct answers: 1, 4 and 5

Tuberculosis occurs in 1.2–6.4% and up to 15% of solid-organ transplant (SOT) recipients in developed and endemic countries respectively. The exact prevalence of tuberculosis (TB) in liver transplant recipients (LTR) in India is not known. Data from centre estimates it to be 3.5%.⁴⁰ The most common form of acquisition of tuberculosis after transplantation is the reactivation of latent infection in patients with previous exposure.⁴¹ Re-infection is also common in endemic areas with high transmission rates. The relative importance of reactivation and re-infection has been debated and ongoing re-infection is responsible for a significant proportion of new infections in high-prevalence areas. Donor-transmitted tuberculosis (both living and cadaveric) accounts for about 4% of all LT-TB.⁴² The diagnosis may be delayed due to the increased frequency of atypical manifestations and extra-pulmonary tuberculosis (60%). Therapeutic dilemmas arise due to drug interactions between rifampicin and tacrolimus. Post-transplant TB may be a life-threatening disease with mortality ranging from 18% to 32%, much higher than in the general population.⁴³ For these reasons it is recommended to screen potential LTRs for latent TB. However, TST is unreliable test for latent TB, especially in cirrhotics due to anergy. The role of IFN-γ release assays (IGRA) in predicting latent TB in LTRs is under study. The value of a single IGRA is not better than tuberculosis skin test (TST) in full form (LTRs), IGRAs cannot rule out active tuberculosis and tuberculosis has been reported in recipients with negative IGRA results.^44–46 The value of serial testing of IGRA's to predict tuberculosis remains to be explored in LTRs.⁴⁰ Prophylaxis with isoniazid in TST positive patients is also controversial. Only patients with recent TST conversion, recipients of donors with evidence of tuberculosis or recipients reporting exposure to patients with pulmonary tuberculosis will probably benefit with prophylaxis.⁴⁰ Prophylaxis is not useful in patients with positive TST due to prior infection. Isoniazid related hepatitis occurs in 6–20% of LTRs and isoniazid is poorly tolerated.⁴² Completion rates for 9 months prophylaxis therapy are less than 50%. Hence, not all potential LTRs with TST positivity should be started on prophylaxis therapy.⁴⁰

7. Correct answers: 1, 3 and 4

Portal hypertension develops due to increased intrahepatic vascular resistance and increased portal blood flow. Increased intrahepatic vascular resistance is due to combination of mechanical factors like architectural disruption of the sinusoids due to fibrosis and nodule formation and dynamic factors like vasoconstriction of intrahepatic vessels. Intrahepatic vasoconstriction might contribute 10–30% of the increase in portal resistance or even more according to recent studies.^48,49 Increased portal blood flow occurs due to splanchnic vasodilatation due to an overproduction of endogenous vasodilator like nitric oxide (NO) and a decrease in vascular reactivity to vasoconstrictors. In addition to these mechanisms, sinusoidal remodelling and angiogenesis also contribute to development of portal hypertension (PHTN). Sinusoidal remodelling encompasses the recruitment of full form (HSCs) to the sinusoidal walls and activation of HSCs with extension of their tentacle-like structures which encircle the vascular lumen and adjacent endothelial cells.⁴⁹ Recently, platelet derived growth factor (PDGF) signalling has been found to be crucial for angiogenesis and sinusoidal remodelling.⁵⁰ It acts as a potent mitogen for HSCs and its expression is up-regulated in chronic liver disease.⁵¹ Angiogenesis is regulated by full form VEGF as well as PDGF, and causes increased portal blood flow, as well as the development of porto-systemic collaterals. Most of the current therapies for PHTN like β blockers, terlipressin, somatostatin and octreotide focus on reducing splanchnic vasodilation and portal blood flow. Traditional therapies for reduction in hepatic vasoconstriction like NO and endothelin receptor antagonists are either not clinically beneficial or limited by side effects.⁴⁷ Newer potential therapies include simvastatin, angiotensin receptor blockers, toll-like receptor (TLR) 4 blockers and anti-angiogenic agents. Simvastatin acts by increasing endothelial full form NO synthase phosphorylation leading to increased NO production within the liver to cause intrahepatic vasodilatation.⁵²

8. Correct answers: 1 and 5

Dietary changes and increase physical activity remain the backbone of treatment regimens for patients with full form NASH. Evidence suggests that a loss of 5–10% from baseline body weight improves insulin resistance, aminotransferases, and histology.⁵³ However, a majority of patients have difficulty achieving and maintaining desirable weight loss. In one study on 39% patients were able to achieve target weight loss of 7% that was required to produce histological imptovement.⁵⁴ Orlistat is the best studied weight loss drug in NAFLD and cause significant weight loss in NAFLD patients. However, in a randomized controlled trial (RCT) of orlistat versus hypocaloric diet there was no significant difference in weight loss or any of the liver histological parameters between treatment groups. Only 32% achieved target weight loss despite orlistat.⁵⁵ The best dietary composition for NAFLD is not clear. Both low-fat⁵⁶ and low-carbohydrate diets⁵⁷ have shown to be beneficial in NAFLD. In one RCT, 11 weeks of a low-carbohydrate caloric restriction induced comparable weight loss, and hepatic and intra-abdominal fat reduction, but improved hepatic insulin sensitivity by nearly threefold compared with a high-carbohydrate isocaloric reduction suggesting that caloric restriction is the most important goal for improving hepatic steatosis in NAFLD, but carbohydrate restriction may further benefit glucose metabolism in glucose-intolerant patients.⁵⁸ A recent meta-analysis has shown that bariatric surgery like gastric bypass or banding improves steatosis, steatohepatitis as well as fibrosis.⁵⁹

9. Correct answers: 2, 3 and 4

Non-invasive techniques such as ultrasound, CT, MRI, and proton magnetic resonance spectroscopy (¹H-MRS) can detect hepatic steatosis, but cannot reliably distinguish simple steatosis from NASH or grade degree of fibrosis.⁶⁰ Biopsy is considered the gold standard for detection of NASH and grading of fibrosis. Many non-invasive tests are available for detection of fibrosis and NASH but only few have been validated. Combination of various biomarkers of hepatocyte apoptosis, inflammation, and oxidative stress has been used to form predictive scores for diagnosis of NASH. Cytokeratin-18 (CK-18), a marker of apoptosis has been validated in several studies, and a recent meta-analysis found a poled area under ROC curve (AUROC), sensitivity and specificity of 0.82, 0.78 and 0.87 respectively.⁶¹ Combination of CK-18 with other markers has also been used to develop models to predict NASH, including NASH diagnostics (total CK-18, cleaved CK-18, adiponectin and resistin) and NASH diagnostic panel (diabetes mellitus, gender, body mass index, triglycerides, total CK-18, and cleaved CK-18).⁶⁰ Elevated ferritin levels (>1.5 times upper limit of normal) has also been shown to be associated with the diagnosis of NASH and higher NAFLD activity score (NAS).⁶² Transient elastography (TE) has been validated for use in NAFLD patients, with failure rates around 5% due to obesity. Stepwise increase in liver stiffness with increasing histological fibrosis has been demonstrated in NAFLD patients. The AUROC of TE for F3 or higher and F4 disease was 0.93 and 0.95, respectively, and was significantly higher than that of serum markers of fibrosis.⁶³

10. Correct answers: 1 and 4

The most cost-effective strategy for identifying cases of HH in the is phenotypic screening with standard serum iron markers with genotypic confirmation of homozygosity in those with markers of iron overload.⁶⁴ Using a cut-off of >45% for transferring saturation, 98–100% cases homozygote for C282Y mutation could be detected.^65,66 However, specificity was only 44%, thus confirmation is required using genotype analysis is required. Since the penetrance of the C282Y mutation is variable, gene mutation analysis should be done regardless of the transferrin saturation or serum ferritin in first-degree relatives of a HH case. Mutation analysis in the spouse allows for assessment of the genotypic status of the children. If the spouse has no C282Y mutation, the offspring can only be heterozygous but if the spouse is a heterozygote, the child has a 50% chance of being homozygous. Because organ damage is virtually unknown in HH before adult life, evaluation of first-degree relatives can be postponed until about 20 years of age.⁶⁴ Liver biopsy is not required for diagnosis, but should be performed in patients of HH over 40 years of age or having elevated liver enzymes or serum ferritin >1000 mg/ml to evaluate for severity of liver disease. The hepatic iron index (HII) in excess of 1.9 mmol/g/y is strong evidence for HH but is not considered essential for diagnosis of HH as upto 15% patients do not have value above 1.9.⁶⁷ Patients of secondary iron overload due to dyserythropoietic or haemolytic anaemia may have HII comparable with that seen in HH. Institution of phlebotomy therapy before cirrhosis and/or diabetes develop significantly reduces the morbidity and mortality.⁶⁸ Therefore, preemptive treatment of those at risk is advocated. This includes treatment of asymptomatic individuals with homozygous HH and markers of iron overload. In symptomatic patients treatment also mitigate organ damage and improves symptoms like malaise, fatigue, skin pigmentation, insulin requirements in diabetes and abdominal pain. Regular phlebotomy with a target serum ferritin concentration below 50 ng/ml is advocated, but anaemia should be prevented by maintaining haematocrit above 37%. Iron-chelating drug deferoxamine is used in patients with HH and cardiac manifestations or in patients who cannot tolerate phlebotomy due to anaemia.