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The approach to the child with liver disease should be based on an accurate clinical history and a thorough physical examination. Investigating the liver relies on a multidisciplinary approach involving clinical chemistry, hematology, immunology, imaging studies, endoscopy, histopathology, and microbiology. Mutational analyses for many genetic liver diseases are now available. This chapter will outline the basic laboratory assessment of the liver and main disease categories and summarize specialized laboratory investigations which identify the underlying diagnosis.

Baseline Investigations: Biochemical Liver Function Tests

The main functions of the liver include synthesis (albumin, coagulation factors, bile acids), metabolism (carbohydrate, lipid, protein), degradation/detoxification, and excretion (Table 3.1). Biochemical liver function tests (Table 3.2) reflect the severity of hepatic dysfunction but rarely provide diagnostic information on individual diseases.

Table 3.1 Functions of the liver
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Table 3.2 Baseline and initial investigations for the evaluation of liver function
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Bilirubin: Conjugated bilirubin is nearly always elevated in liver disease [1]. The presence of bilirubin is always abnormal if detected in a fresh urine specimen.

Aminotransferases are intracellular enzymes, which are present in liver, heart, and skeletal muscles. Increases in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indicate hepatic necrosis irrespective of etiology (Table 3.2). ALT is more liver specific than AST but has a longer plasma half-life (approximately 24 h). A rise in AST is an early indication of liver damage and is a useful marker of rejection post-liver transplant. Elevated aminotransferases are often the first indication of the development of nonalcoholic fatty liver disease (NAFLD) in an obese child. Elevated aspartate and/or alanine aminotransferases are also found in muscular dystrophy and this diagnosis should be considered if there are no other signs of liver disease. These enzymes, however, may be normal in compensated cirrhosis.

Alkaline phosphatase is found in the liver, kidney, bone, placenta, and intestine. In pediatric liver disease, a raised alkaline phosphatase indicates biliary epithelial damage, malignant infiltration, cirrhosis, rejection, or osteopenia secondary to vitamin D deficiency. In a growing child, however, the potential contribution from bone makes alkaline phosphatase measurement less specific for liver pathology.

Gamma-glutamyl transpeptidase (GGT) is present in biliary epithelia and hepatocytes and also in the cell membrane of many other human organs, including kidney, pancreas, spleen, brain, breast, and small intestine. An elevated GGT is not specific for liver disease. In addition, the reference range is age related, with higher levels in neonates (up to 385 IU/l). It is elevated in many forms of liver damage. However, GGT does not increase in the serum of patients with bone disease or children with active bone growth, thus helpful in confirming the liver origin of a raised alkaline phosphatase. It may be normal in certain forms of intrahepatic cholestasis (progressive familial intrahepatic cholestasis 1 and 2; PFIC 1 & 2) [2].

The most useful tests of liver “function” are plasma albumin concentration and coagulation time. In the absence of excessive urinary or gastrointestinal loss or prolonged starvation, a low serum albumin, which has a half-life of 20 days, indicates chronicity of liver disease. Abnormal coagulation, especially prothrombin time (PT) after vitamin K deficiency is ruled out, indicates significant hepatic dysfunction, either acute or chronic. Fasting hypoglycemia in the absence of other causes (e.g., hypopituitarism or hyperinsulinism) indicates poor hepatic function and is a guide to prognosis in acute liver failure. If these baseline investigations suggest hepatic dysfunction, then more specific investigations for metabolic disease are appropriate to consider [3–5] (Table 3.3).

Table 3.3 Laboratory assessment in chronic liver disease
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Second-Line Investigations

Hepatic dysfunction may be secondary to sepsis, particularly urinary sepsis, inborn errors of metabolism, or endocrine disorders. It is usual to exclude sepsis by performing bacterial culture of the urine and/or blood and cerebrospinal fluid cultures if appropriate (Tables 3.3 and 3.4).

In neonates, hypopituitarism may be difficult to exclude as thyroid function tests may be equivocal or in the low normal range. It is useful to perform a 09.00 h cortisol level at the same time as measuring free thyroxine and thyroid-stimulating hormone (TSH) [6].

If the infant is unwell, or has evidence of acute liver failure, galactosemia and tyrosinemia should be excluded (see below). Urea cycle defects should also be considered particularly if the serum ammonia is raised.

Alpha-1-antitrypsin deficiency is the most common inherited metabolic liver disease and should always be excluded, regardless of age. As α-1-antitrypsin is an acute-phase protein, it is necessary to measure both concentration and phenotype in order to differentiate between normal and an acute-phase response in the setting of homozygous or heterozygous deficiency.

Although cystic fibrosis is a rare cause of liver disease in the neonatal period, it should be considered in the differential diagnosis of neonatal liver disease and excluded by performing an immunoreactive trypsin test, a sweat test, and mutational analysis if either is positive.

Wilson disease rarely presents before the age of 3 years but may mimic any form of liver disease and should always be excluded in older children [7]. An autoimmune screen and immunoglobulin levels should detect 75 % of children with autoimmune hepatitis.

Serum cholesterol is usually elevated in children with severe cholestasis (e.g., Alagille syndrome, biliary atresia). In contrast, low or normal cholesterol is characteristic of bile acid transport disorders or terminal liver disease.

Plasma ammonia and amino acids (particularly phenylalanine, tyrosine, and methionine) may be raised in either acute or chronic liver failure and are nonspecific indications of hepatic dysfunction. Primitive hepatic cells synthesize α-fetoprotein. The levels are highest in the newborn (>1,000 mg/l) and fall in the first few months of life. It may be a useful screening test in the diagnosis of tyrosinemia type I and hepatoblastoma or for detection of hepatocellular carcinoma in chronic carriers of hepatitis B and C. The α-fetoprotein level can be as high as 100,000 mg/l in hepatoblastoma [8].

Neonatal Liver Disease

Most infants with liver disease present in the neonatal period with persistent jaundice. Although physiologic jaundice is common in neonates, infants who develop severe or persistent jaundice should be investigated to exclude hemolysis, sepsis, or underlying liver disease. Neonatal jaundice that persists beyond 14 days in term infants and 21 days in preterm infants should always be investigated, even in breast-fed babies [1]. It is also necessary to establish whether the jaundice is due to an increase in conjugated or unconjugated hyperbilirubinemia.

Unconjugated hyperbilirubinemia: Common causes include ABO and rhesus incompatibilities, breast-milk jaundice, sepsis, Gilbert syndrome, and rarely, Crigler–Najjar type I or II (Table 3.5).

Table 3.4 Age-specific investigations in chronic liver disease
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Conjugated hyperbilirubinemia: A rise in conjugated bilirubin always signifies an underlying liver condition and warrants further assessment (Table 3.6) [1]. It is important to exclude surgical disorders such as biliary atresia in infants with neonatal cholestasis as early surgery is associated with a better outcome (Table 3.7) [9, 10]. Similarly, bacterial infections and metabolic conditions have improved outcomes with early identification and treatment and hence warrant rapid investigation. Although not usually posing a diagnostic dilemma, the successful management of preterm infants as young as 25 weeks’ gestation has increased the number of children treated with parenteral nutrition (PN) and a consequential rise in referrals of these infants with persistent jaundice [11]. Other conditions to be considered that can present as neonatal cholestasis are listed in Table 3.8 [1, 12].

Table 3.5 Common and uncommon causes of unconjugated hyperbilirubinemia in infancy
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Table 3.6 Laboratory assessment of the cholestatic infant
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Table 3.7 Assessments of infants (2 weeks–6 weeks of age) suspected of biliary atresia
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Fig. 3.1
figure 1

A typical appearance of pale stool

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Liver Disease in Older Children

Liver disease in children older than 6 months may be acute or chronic. As in infancy, inherited disorders need to be excluded (Table 3.9), but jaundice may not be a prominent feature. Acute or chronic liver disease may be due to infection, autoimmune disease, drug-induced hepatitis, and metabolic diseases (Table 3.9).

Table 3.8 Differential diagnosis of infantile conjugated hyperbilirubinemia when biliary atresia has been excluded
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Acute Liver Disease

Underlying causes and clinical presentation depends on the age, but the following clinical features are common: a prodrome of malaise, lethargy, and anorexia, and nausea, vomiting, or diarrhea. There may be weight loss, abdominal discomfort, tender hepatomegaly, splenomegaly, ascites (rarely, except for acute Budd–Chiari), rash, or joint pains. It is noteworthy that jaundice is not always present.

The differential diagnosis of acute hepatitis in older children includes (Table 3.9) [13, 14]: viral hepatitis A, B, C, and E, sero-negative hepatitis, autoimmune hepatitis, drug-induced hepatotoxicity, and metabolic liver disease especially Wilson disease.

Important causes of neonatal liver failure include viral infections, metabolic liver disease, and ischemic causes (Table 3.10) [15].

Table 3.9 Causes of acute liver disease and failure in older children
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Chronic Liver Disease

  • Chronic liver disease is frequently asymptomatic but detected through other analyses, such as incidental detection of abnormal liver enzymes or hepatomegaly

  • Family screening for hepatitis B/C or metabolic disorders (Wilson disease)

  • Transfusion recipient following diagnosis of donor infection

  • Coexistent disease, e.g., inflammatory bowel disease and celiac disease

  • Recipient of a known toxic agent, e.g., methotrexate

When symptomatic children may present with:

  • Intermittent fatigue, anorexia, and weight loss

  • Abdominal discomfort

  • Variable or fluctuating jaundice with pruritus and pale stools

  • Hematemesis or melena from variceal bleeding – especially with portal hypertension

Liver Biopsy and Histopathology

Table 3.10 Causes of neonatal acute liver failure
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The diagnosis of most chronic liver diseases requires histological confirmation [16]. An aspiration technique, using a Menghini needle (or disposable variant), has a complication risk of 1:1,000 liver biopsies and may be performed under sedation with local anesthesia. In fibrotic or cirrhotic livers, the use of a Tru-Cut needle, with a cutting-edge beveled end, may be necessary. Transjugular liver biopsies, in which the liver is biopsied through a special catheter passed from the internal jugular vein into the hepatic veins, are now possible for children as small as 6 kg and are a safer way to perform a biopsy if coagulation times remain abnormal despite support (prothrombin time [PT] >5 s prolonged over control value) or for those with large ascites [17]. The complications of this potentially dangerous procedure (see below) are much reduced if performed in expert hands, in specialized units, under controlled conditions [18]. It is essential to be aware of the absolute and relative contraindications of liver biopsy. Biopsy specimens should be obtained for routine histopathology and can be analyzed for microbiology, electron microscopy, immunohistochemistry, and copper (if appropriate) and snap frozen in liquid nitrogen for enzymatic or metabolic investigations. As the interpretation of the histology may be difficult and requires considerable specialist expertise, and tissue preparation for other analyses requires special handling, coordination with the liver pathologist before tissue acquisition is advisable.

In experienced facilities and with careful patient selection, it is possible to carry out a liver biopsy as a day procedure [19, 20]

Complications of Percutaneous Liver Biopsy

Although uncommon, the main complication of percutaneous liver biopsies is bleeding. Subclinical bleeding (as evident on ultrasound imaging) is common and intrahepatic and subcapsular hematomas with no hemodynamic compromise are seen in up to 23 % of patients [21]. Significant nonfatal bleeding (as seen with evidence of active bleeding, shock, or a hemoglobin drop of 2.0 g/l) occurs more frequently in children than adults. In adults significant hemorrhage occurs in 0.3–0.5 % of cases, while bleeding requiring transfusion is seen in up to 2.8 % of children [22]. Evidence of persistent bleeding following liver biopsy despite medical support and blood transfusion warrants urgent hepatic angiography and embolization or surgery.

Other complications include:

  • Pneumothorax or hemopneumothorax

  • Infection (particularly if the biopsy is combined with another procedure, e.g., dental extraction)

  • Perforation of the gall bladder or bile ducts leading to biliary peritonitis

Adequate monitoring of vital signs post biopsy is essential to detect complications such as hemorrhage or infection [22].

Table 3.11 Specific investigations for metabolic liver diseases
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Metabolic Investigations (Table 3.11)

Many inborn errors of metabolism present with hepatomegaly and/or liver disease. It is essential to screen for these diseases as part of the investigation of liver disease in neonates and in older children

Bone Marrow Aspiration

Bone marrow aspiration may be useful in infants with undiagnosed neonatal hepatitis and hepatomegaly and splenomegaly, in order to exclude Niemann–Pick type C, or at any age if a storage disorder is suspected and genetic testing unavailable.

Skin Biopsy with Fibroblast Culture

This procedure can be useful in diagnosing inborn errors of metabolism (e.g., Niemann–Pick type A, B, or C or tyrosinemia type I) when genetic testing is not available (Table 3.11).

Genetic Tests (Chromosome and DNA)

With the rapid development of molecular techniques for diagnosis and detection of genetic diseases, samples for DNA analysis and/or chromosomes from both child and parent are essential and now possible for many genetic conditions affecting the liver.

Neurophysiology

Electroencephalography (EEG) is mostly used in the assessment of hepatic encephalopathy. It will identify abnormal rhythms secondary to encephalopathy due to either acute or chronic liver failure or drug toxicity such as posttransplant immunosuppression, but findings are frequently nonspecific. EEG may also be of value in verifying brain death as a flat EEG in the absence of sedation is an indication for withdrawal of therapy.

Ophthalmology (Table 3.12)

Table 3.12 Ophthalmic lesions in liver disease
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A number of inherited conditions have associated ophthalmic lesions (e.g., posterior embryotoxin in Alagille syndrome, Kayser–Fleischer rings in Wilson disease), and thus, ophthalmological examination should be part of the assessment process when these conditions are suspected. Additionally, children with Alagille syndrome have a higher-than-normal incidence of benign intracranial hypertension, and thus, annual fundoscopy for papilledema is essential [23].

Endoscopic Retrograde Cholangiopancreatography (ERCP)

This procedure is invaluable for the assessment of extrahepatic biliary disease in older children (e.g., choledochal cysts, primary sclerosing cholangitis) or for the assessment of chronic pancreatitis. It involves an endoscopic technique where a fiberoptic duodenoscope is passed into the first part of the duodenum, the ampulla of Vater is identified, the pancreatic and biliary ducts are cannulated, and radiological contrast is injected. The technique has an 80 % success rate in skilled hands. Although this technique should be of value in the differential diagnosis of neonatal cholestasis, technical difficulties in the cannulation of bile ducts in small infants may provide equivocal information. Recently, in some centers, the diagnostic value of ERCP has been superseded by magnetic resonance imaging (MRI) via performance of a magnetic resonance cholangiopancreatography (MRCP) which is noninvasive. Limitations of MRCP due to experiential lack of sensitivity mean ERCP is still valuable as a diagnostic tool, and ERCP also retains an important role in therapy [24].

Molecular Biology

The development of molecular biology has revolutionized methodology for many complex diagnostic procedures, transforming many techniques into routine laboratory procedures [25], particularly in screening for rare neonatal diseases [26]. Progress in identifying specific genes and DNA sequencing has made possible the diagnosis of many inborn errors of metabolism and inherited disease (e.g., Alagille syndrome, Wilson disease, tyrosinemia type I) and led to the identification of specific mitochondrial disorders.

Advances in methodology for gene cloning and molecular cloning methods have been helpful in identifying viruses such as hepatitis C and G [27], while the polymerase chain reaction has been used to diagnose active infection and monitor patients with many different viral diseases, such as hepatitis C, cytomegalovirus (CMV), and Epstein–Barr virus (EBV). Diagnosis for autoimmune disorders has improved, with specific assays that use recombinant protein antigens (e.g., antinuclear antigens and liver–kidney microsomal antibodies). The rapid development of molecular techniques is certain to lead to further improvements in diagnostic methods and to a better understanding of pediatric liver disease.