Liver Function Tests

Etiology and Epidemiology

Elevated liver function tests are found in approximately 8% of the general population. These elevations may be transient in patients without symptoms, with up to 30% of elevations resolving after three weeks. Thus, care should be taken when interpreting these results to avoid unnecessary testing.[5] A borderline AST and/or ALT elevation is defined as less than 2 times the upper limit of normal (ULN), a mild AST and/or ALT elevation as 2 to 5 times ULN, moderate AST and/or ALT elevation 5 to 15 times ULN, severe AST and/or ALT elevation greater than 15 times ULN, and massive AST and/or ALT greater than 10,000 IU/l.The magnitude of AST and ALT elevation varies depending on the cause of hepatocellular injury.[6]

Differential Diagnosis Based on Elevated LFTs

Hepatocellular pattern: Elevated aminotransferases out of proportion to alkaline phosphatase

• ALT-predominant:  Acute or chronic viral hepatitis, steatohepatitis, acute Budd-Chiari syndrome, ischemic hepatitis, autoimmune, hemochromatosis, medications/toxins, autoimmune, alpha1-antitrypsin deficiency, Wilson disease, Celiac disease

• AST-predominant: Alcohol-related, steatohepatitis, cirrhosis, non-hepatic (hemolysis, myopathy, thyroid disease, exercise)

Cholestatic pattern: Elevated alkaline phosphatase +gamma glutamyl transferase + bilirubin out of proportion to AST and ALT

• Hepatobiliary causes: Bile duct obstruction, primary biliary cirrhosis, primary sclerosing cholangitis, medication-induced, infiltrating diseases of the liver (sarcoidosis, amyloidosis, lymphoma, among others), cystic fibrosis, hepatic metastasis, cholestasis

• Non-Hepatic causes: Bone disease, pregnancy, chronic renal failure, lymphoma or other malignancies, congestive heart failure, childhood growth, infection, or inflammation

Pathophysiology

Components of Liver Function Test

Hepatocellular Labs

Aminotransferase includes AST and ALT. They are markers of hepatocellular injury. They participate in gluconeogenesis by catalyzing the transfer of amino groups from aspartic acid or alanine to ketoglutaric acid to produce oxaloacetic acid and pyruvic acid, respectively. AST is present as cytosolic and mitochondrial isoenzymes and is found in the liver, cardiac muscle, skeletal muscle, kidneys, brain, pancreas, lungs, leucocytes, and red cells. It is not as sensitive or specific for the liver as ALT and elevation in AST may be seen as secondary to nonhepatic causes as well. AST activity in neonates and infants is approximately twice that in adults, but these decline to adult levels by approximately six months.[7]

ALT is a cytosolic enzyme that is found in high concentrations in the liver. The half-life of ALT is approximately 47 ± 10 hours. ALT is usually higher than AST in most types of liver disease in which the activity of both enzymes is predominantly from the hepatocyte cytosol. Hepatocellular injury and not necessarily cell death triggers the release of these enzymes into circulation. Both AST and ALT values are higher in normal males than females.[8]They also correlate with obesity with a normal reference range higher in those with higher body mass index.[9]

Cholestasis Labs

Alkaline phosphatase is part of a family of zinc metalloenzymes that are highly concentrated in the microvilli of the bile canaliculus as well as several other tissues (e.g., bone, intestines, and placenta).[1] There are four isozymes: placental ALP or hPLALP (human placental ALP), germ cell ALP (GCALP or PLALP-like),  intestinal ALP (IALP), and tissue-nonspecific ALP (TNALP). Of these four, PLALP and GCALP are the most heat stable at 65 C, and the bone ALP component of TnALP is the least. In healthy, non-smoking individuals, the PLALP and GCALP represent less than 1% of total ALP activity in the serum.[10] 

A condition that can result in significantly increased plasma ALP is benign transient hyperphosphatasemia. Originally described in infants, transient hyperphosphatasemia can also occur in adults and during pregnancy. There is a marked rise in ALP, often to several thousand IU/L, which usually indicates significant pathology. It is, however, a benign condition with a return to normal of the ALP in 6 to 8 weeks. Transient hyperphosphatasaemia is associated with concurrent infections in over 60% of cases, particularly GIT infections. There is a characteristic pattern on polyacrylamide gel electrophoresis, with the normal pattern of isoenzymes being accompanied by variant forms that react with neuraminidase. It is believed to be due to changes in carbohydrate side chains causing failure of recognition by receptors and reduced clearance, thus prolonging half-life.[11] 

Glycoprotein gamma-glutamyltransferase (GGT) is located on membranes of cells with high secretory or absorptive activities. Its primary function is to catalyze the transfer of a gamma-glutamyl group from peptides to other amino acids. It is also abundant in many other sources of the body (kidney, pancreas, intestine, prostate, testicles, spleen, heart, and brain) but is more specific for biliary disease when compared to alkaline phosphatase because it is not present in bone. Serum GGT shows electrophoretic mobility and lectin-affinity reaction identical to the liver enzyme but different from GGT from the kidney, urine, and pancreas.GGT levels are reported to be increased by an average of 12-fold in obstructive liver disease compared to ALP, which increased only 3-fold, so GGT is slightly more sensitive than ALP in this regard.GGT activity level in children may be a reliable index of bile duct damage. It is a useful indicator in separating the two forms of idiopathic cholestasis, with or without bile duct involvement. In infants diagnosed with biliary atresia and managed surgically, the GGT levels stay high in the blood if the infant is breastfed. This is due to the high level of GGT in human breast milk for at least four weeks postpartum.[12] 

There is a relationship between plasma GGT activity and weight, with values being 50% higher in individuals with a BMI  greater than 30 kg/m². This is believed to be due to fat deposition in the liver (steatosis) in obese subjects. Steatosis with a raised plasma GGT also occurs in diabetes mellitus, non-alcoholic steatohepatitis, and non-alcoholic fatty liver disease. Any liver disease that results in fibrosis and/or cirrhosis, such as alcoholic cirrhosis, PBC, PSC, hemochromatosis, α1-antitrypsin deficiency, and Wilson disease, will cause a raised plasma GGT. Space-occupying lesions, including malignancy (HCC or metastases secondary to malignancy elsewhere in the body), and granulomatous disease, for example, sarcoidosis and TB, are also associated with a raised plasma GGT.5′-Nucleotidase (5′NT) is associated with the canalicular and sinusoidal plasma membranes. Its function is undefined. 5′NT is also found in the intestine, brain, heart, blood vessels, and endocrine pancreas. Serum levels of 5′NT are unaffected by sex or race, but age affects the level; values are lowest in children and increase gradually, reaching a plateau at approximately age 50 years. As with GGT, the primary role of the serum 5′NT level is to identify the organ source of an isolated serum alkaline phosphatase elevation. The 5′NT level is not increased in bone disease but primarily in hepatobiliary disease. LDH is commonly included in biochemical liver panels but has poor diagnostic specificity for liver disease. Markedly increased LDH levels are observed in hepatocellular necrosis, shock liver, lymphoma, or hemolysis associated with liver disease.[13] 

Bilirubin is the end product of heme catabolism, with 80% derived from hemoglobin. Unconjugated bilirubin is transported to the liver loosely bound to albumin. Bilirubin is water-insoluble and cannot be excreted in the urine. Bilirubin that is conjugated is water-soluble and appears in the urine. It is conjugated in the liver to bilirubin glucuronide and subsequently secreted into bile and the gut, respectively.[14]

Synthetic Function Tests

Albumin is synthesized by the hepatic parenchymal cells at a rate dependent on colloidal osmotic pressure and dietary protein intake. The rate of albumin synthesis is also subject to feedback regulation determined by the plasma albumin concentration. Maintenance of plasma albumin concentrations can be achieved with only 10% of normal hepatocyte mass. The half-life of albumin is 21 days. Traces of albumin can be found in almost all extracellular body fluids. Little is lost from the body by excretion.[15] It is catabolized in various tissues, which are taken up by cells by pinocytosis. Its constituent amino acids are released by intracellular proteolysis and returned to the body pool. With any liver disease, there is a fall in serum albumin, reflecting decreased synthesis. If liver function is normal and serum albumin is low, this may reflect poor protein intake (malnutrition) or protein loss (nephrotic syndrome, malabsorption, or protein-losing enteropathy).[16]

Prothrombin time (PT) measures the rate of conversion of prothrombin to thrombin. Except for factor VIII, all other coagulation factors are synthesized by the liver. Prothrombin time requires factors II, V, VII, and X, and, as these are made in the liver, the liver's function is crucial in coagulation. Suppose the synthetic function of the liver is normal and prothrombin time is delayed. This may indicate treatment with warfarin, consumptive coagulopathy (eg, disseminated intravascular coagulopathy), or vitamin K deficiency.[17]

Serological Tests

Liver-related autoantibodies are crucial for correctly diagnosing and classifying autoimmune liver diseases, namely autoimmune hepatitis types 1 and 2 (AIH-1 and 2), primary biliary cirrhosis (PBC), and the sclerosing cholangitis variants in adults and children.AIH-1 is specified by anti-nuclear antibody (ANA) and smooth muscle antibody (SMA). AIH-2 is specified by antibody to liver kidney microsomal antigen type-1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1). SMA, ANA, and anti-LKM antibodies can be present in de-novo AIH following liver transplantation.[18]PBC is specified by antimitochondrial antibodies (AMA) reacting with enzymes of the 2-oxo-acid dehydrogenase complexes (chiefly pyruvate dehydrogenase complex E2 subunit) and disease-specific ANA mainly reacting with nuclear pore gp210 and nuclear body sp100. Sclerosing cholangitis presents in at least two variants; first, the classical primary sclerosing cholangitis (PSC) mostly affects adult men wherein the only (and non-specific) reactivity is an atypical perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA), also termed perinuclear anti-neutrophil nuclear antibodies (p-ANNA) and second the childhood disease called autoimmune sclerosing cholangitis (ASC) with serological features resembling those of type 1 AIH.[19]

Secondary Biochemical Liver Tests

Alpha-fetoprotein (AFP) measurements are used as a tumor marker for the detection and monitoring of primary hepatocellular malignancies, such as hepatoblastoma and HCC. Hepatoblasts produce alpha-fetoprotein, which is why it is raised in the regenerating liver, particularly in chronic viral hepatitis.[20]

Carbohydrate deficient transferrin is a high-specificity test for detecting excess alcohol intake as a cause of liver damage. The carbohydrate antigen CA19-9 is useful in monitoring the activity of the autoimmune disease PSC, which often progresses to a tumor of the bile ducts or cholangiocarcinoma.[21] Measurement of serum ferritin can be useful in identifying hemochromatosis, but ferritin is a positive acute phase reactant, so it is raised in many illnesses as well as being released from damaged hepatocytes in acute hepatic failure.[22]

Specimen Requirements and Procedure

The serum is the specimen of choice. Consider all plasma or serum specimens potentially positive for infectious agents, including HIV and the hepatitis B virus. All specimens should be handled with standard precautions and sent to the lab immediately for processing. Separated serum or plasma should not remain at +15 C to +30 C longer than 8 hours. If assays are not completed within 8 hours, serum or plasma should be stored at +2 C to +8 C. If assays are not completed within 48 hours, or the separated sample is to be stored beyond 48 hours, samples should be frozen at –15 C to –20 C. Frozen samples should be thawed only once. Analyte deterioration may occur in samples that are repeatedly frozen and thawed.[23]

Testing Procedures

Liver function tests are performed on semi-automatic or fully automated analyzers, which are based on the principle of photometry. Photometry is the measurement of light absorbed in the ultraviolet (UV) to visible (VIS) to infrared (IR) range. This measurement is used to determine the amount of an analyte in a solution or liquid. Photometers utilize a specific light source and detectors that convert light passed through a sample solution into a proportional electrical signal. These detectors may be photodiodes, photoresistors, or photomultipliers. Photometry uses Beer–Lambert’s law to calculate coefficients obtained from the transmittance measurement. A correlation between absorbance and analyte concentration is then established by a test-specific calibration function to achieve highly accurate measurements.[24]

Interfering Factors

Hemolysis, icterus, and lipemia are the most common specimen integrity issues that can interfere with laboratory tests and may lead to erroneous results and interpretations and, ultimately, to inappropriate medical decisions. Oxygenated and deoxygenated hemoglobin have slightly different absorbance spectra but show maximal absorbance around 415 nm, with significant absorbance between 320 and 450 nm and 540 and 580 nm. Colorimetric assays that use absorbance measurements in one or more of these ranges are, therefore, susceptible to interference. Examples include iron, lipase, albumin, and g-glutamyl transferase.

Alkaline phosphatase assays are susceptible to negative interference because alkali denaturation of hemoglobin may cause a negative offset in absorbance readings. Icterus exerts effects on chemistry tests primarily through spectrophotometric and chemical interferences. Bilirubin absorbs light between 400 and 540 nm, with a peak around 460 nm. Colorimetric assays taking primary or secondary absorbance measurements at these wavelengths may be affected. The unconjugated and conjugated forms of bilirubin may exert different effects on certain assays. Conjugated bilirubin has been found to cause a greater degree of interference with most assays.

Lipemia causes light scattering, differential partitioning of analyte between the polar and aqueous phases of the sample, and interaction of the lipoprotein particles with assay reagents may all cause interference in results. Lipaemia causes light scattering across the visual spectrum (300 to 700 nm). Colorimetric assays taking absorbance readings at the shorter wavelengths of the visual spectrum are, therefore, most susceptible to interference. As a result, assays that utilize changes in NAD(P)H concentration, measured around 340 nm, are susceptible to lipaemia interference. ALT levels can increase due to certain drugs, which should be avoided before testing.

Drug hepatotoxicity can be nonidiosyncratic (predictable) or idiosyncratic (unpredictable). Also, drug-associated hepatotoxicity can classify as immune-mediated and non-immune-mediated. The incidence of drug-induced liver injury is 19 cases per 100,000 persons. The most common drug causing drug-induced liver injury is amoxicillin/clavulanate.[25][26][27] Diurnal variations in ALT have been observed in both healthy individuals and those with cirrhosis. Up to 45% variation has been seen, with higher values observed in the afternoon.

Other factors which can affect ALT include body mass index (40 to 50% higher with high body mass index) and exercise (20% lower in those who exercise).[28] Metronidazole may interfere with ALT methods because of its relatively high concentration and absorbance near 340 nm.[29]  Following food ingestion, increases of up to 30 U/L may be seen. In patients with blood groups B and O, these increases can persist for up to 12 hours, attributable to the intestinal isoenzyme. Smoking causes elevated levels of PLALP, which return to the normal range after 1 to 2 months of smoking cessation. During growth, due to increased osteoblastic activity, elevated levels of ALP are seen in children and adolescents. The normal reference range levels also increase with age in females. An unexplained high serum ALP result should always be confirmed with a repeat fasting sample. Increases up to two- or three-fold are normal in the third trimester of pregnancy due to the presence of the placental isoenzyme.

Results, Reporting, and Critical Findings

The results of liver function tests should correlate with the initial findings in a complete patient history and physical examination. A thorough review should include important questions regarding the patient's age, past medical history (diabetes, obesity, hyperlipidemia, inflammatory bowel disease, celiac sprue, thyroid disorders, autoimmune hepatitis, acquired muscle disorders, alcohol use disorder, medication use, toxin exposure, and family history of genetic liver conditions (Wilson disease, alpha-1-antitrypsin deficiency, hereditary hemochromatosis).[30]

A review of systems should also include signs and symptoms of chronic liver disease such as jaundice, ascites, peripheral edema, hepatosplenomegaly, gynecomastia, testicular hypotrophy, muscle wasting, encephalopathy, pruritus, and gastrointestinal bleeding. Other tests that help determine the cause of elevated transaminase levels found on a hepatitis panel include fasting lipid levels, hemoglobin A1C level, fasting glucose, complete blood count with platelets, a complete metabolic panel, iron studies, hepatitis C antibody, and hepatitis B surface antigen testing.

Reference ranges for LFTs tend to vary depending on the laboratory. Further, normal reference ranges vary between males and females and may be higher for those with a higher body mass index.[24] A patient's blood test values should be interpreted based on the reference value of the laboratory in which the test is done. It is recommended that each laboratory establish its own reference interval based on its methodology.

• Alanine transaminase: 4 to 36 IU/L
• Aspartate transaminase: 5 to 30 IU/L
• Alkaline phosphatase: 30 to 120 IU/L
• Gamma-glutamyltransferase: 6-50 IU/L
• Bilirubin: 2 to 17 µmol/L
• Direct bilirubin: 0 to 6 µmol/L
• Prothrombin time: 10.9 to 12.5 seconds
• Albumin: 35-50 g/L
• Total protein: 60 to 80 g/L
• Lactate dehydrogenase: 50 to 150 IU/L

Clinical Significance

The levels of LFTs can point to the differentials. Many disease processes have very distinct abnormalities in the liver enzymes. Further investigation is warranted if repeated tests confirm abnormality.

Alcohol

In patients with alcohol use disorder, AST to ALT ratio is generally at least 2:1, showing a high level of AST activity in alcoholic liver disease.[31] Elevated GGT, along with AST, also suggests alcohol abuse. GGT should not be used alone since it is not very specific for alcohol.[32]

Medications

Several medications are known to cause liver damage. Many of these are commonly used in daily practice, including but not limited to NSAIDs, antibiotics, statins, anti-seizure drugs, and drugs for tuberculosis treatment. Acute hepatocellular injury can be seen secondary to several drugs, including but not limited to acetaminophen, allopurinol, NSAIDs, alcohol, anti-tuberculosis medications such as isoniazid, pyrazinamide, rifampin, statins, antifungals such as ketoconazole, antibiotics such as tetracyclines, anti-seizure drugs such as valproic acid and phenytoin, antidepressants such as fluoxetine, antipsychotics such as risperidone and antivirals such as valacyclovir and ritonavir.[25]

Acute cholestasis can be seen secondary to drugs, including anabolic steroids, NSAIDs, tricyclic antidepressants, alcohol, antibiotics such as azithromycin, amoxicillin, nafcillin, rifampin, and trimethoprim-sulfamethoxazole. Long-term use of these agents can also lead to chronic hepatocellular and/or cholestatic liver damage.[26] Methotrexate, the commonly used medication for rheumatoid arthritis and other inflammatory arthritis, can cause a mild transient elevation in LFTs and can also cause permanent liver damage in liver fibrosis and cirrhosis, especially with higher cumulative doses. Liver fibrosis can also be seen as secondary to chronic alcohol intake or methyldopa. Ergot alkaloids can result in ischemic necrosis. Oral contraceptives can result in hepatic venous outflow obstruction (Budd-Chiari syndrome).[33] Herbal medications can also cause an elevation in LFTs.[34]

Viral Hepatitis

Viral illnesses are a common cause of hepatitis and elevation in LFTs. Viral hepatitis B, C, and D can cause chronic hepatitis, while hepatitis A and E cause acute viral hepatitis.[35] Several other viruses, including HIV, Epstein-Barr (EBV), and Cytomegalovirus (CMV), can also cause hepatitis.[36]

Autoimmune Hepatitis

Autoimmune hepatitis is a chronic disease characterized by continuing hepatocellular inflammation, necrosis, and a tendency to progress to cirrhosis. It is more common in young women than men, with a 4:1 ratio. The patient usually presents with high LFTs without apparent cause.[37] These patients can have positive autoantibodies, including antinuclear antibodies, anti-smooth muscle antibodies, anti-liver/kidney microsomal antibodies, and antibodies to the liver antigen.[38]

Hepatic Steatosis and Nonalcoholic Steatohepatitis

Fatty liver disease, aka nonalcoholic steatohepatitis, has recently gained more attention because of its ability to cause chronic hepatic disease and hepatocellular carcinoma (HCC). The typical patient with this disease is overweight, has type II diabetes, or has dyslipidemia and no evidence of clinically significant alcohol use.[39] The AST and ALT are usually both elevated with a ratio of 1:1, with other liver function tests being normal.[40]

Hemochromatosis

Hemochromatosis is abnormal iron accumulation in parenchymal organs, leading to organ toxicity. It is the most common autosomal recessive genetic disorder and the most common cause of severe iron overload. Clinical manifestations include diabetes, liver disease, and cutaneous hyperpigmentation.[41] A raised serum ferritin level usually raises concerns for possible hemochromatosis, but a transferrin saturation greater than 45% is more reliable. HFE mutations (C282Y, H63D) are pivotal for diagnosing hereditary hemochromatosis. Secondary hemochromatosis can also be seen due to increased iron intake.[42]

Wilson Disease

Wilson disease, a rare autosomal-recessive inherited disorder of copper metabolism, is characterized by excess copper deposition in the liver, brain, and other tissues. It is fatal if not recognized and treated early. A low serum ceruloplasmin level is seen in the majority (up to 85%) of the cases.[43] Kayser-Fleischer rings can be a clinical clue but are not always present. The 24-hour urinary copper excretion test is usually abnormal, with more than 100 micrograms of copper excretion in the urine indicating Wilson's disease. A liver biopsy remains the confirmatory test.[44]

Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin deficiency (AATD) is a relatively common yet often undiagnosed genetic condition.[45]Those with AATD are also predisposed to obstructive pulmonary disease and liver disease (e.g., cirrhosis and hepatocellular carcinoma in children and adults). AATD is one of the most common inherited disorders among Caucasians. Its primary manifestation is early-onset panacinar emphysema.[46]

Celiac Disease

Celiac disease is a common gluten sensitivity disorder associated with modest elevations of liver transaminases; screening should be considered in patients with persistently elevated liver enzymes and consists of tissue transglutaminase IgA and serum IgA level or tissue transglutaminase IgA and anti-deamidated gliadin peptide IgG.[47][48]

Thyroid Disorders

Both hypothyroidism and hyperthyroidism have been associated with abnormal liver enzymes, including both hepatocellular and cholestatic patterns of injury, particularly in more severe cases of myxedema and/or thyrotoxicosis. Screening should be considered in patients with a compatible medical history and consist initially of thyroid stimulating hormone and selective testing of free T4 and free/total T3.[49]

Quality Control and Lab Safety

For non-waived tests, laboratory regulations require, at the minimum, analysis of at least two levels of control materials once every 24 hours. Laboratories can assay QC samples more frequently if deemed necessary to ensure accurate results. Quality control samples should be assayed after calibration or maintenance of an analyzer to verify the correct method performance. To minimize QC when performing tests for which manufacturers’ recommendations are less than those required by the regulatory agency (such as once per month), the labs can develop an individualized quality control plan (IQCP) that involves performing a risk assessment of potential sources of error in all phases of testing and putting in place a QC plan to reduce the likelihood of errors.[23] Westgard multi-rules are used to evaluate the quality control runs. In case of any violation of a rule, proper corrective and preventive action should be taken before patient testing is performed.

Enhancing Healthcare Team Outcomes

Liver function tests are one of the most commonly ordered laboratory tests. Mild isolated elevations in LFTs can be seen as normal fluctuations and shall not trigger expensive and extensive workups. However, clinicians shall be aware of various conditions that can lead to an elevation in LFTs. Thorough history taking and physical examination can provide clues to the differential diagnosis.

Drug and medication history are of utmost importance. The nursing team shall help with medication reconciliation. Pharmacists can also assist in identifying potentially hepatotoxic agents. Referral to specialists such as hepatologists may sometimes be indicated. An interprofessional team approach can help identify the underlying etiology with appropriate management.[50] This interprofessional team paradigm for patient care, employing good record keeping and open communication lines between different healthcare disciplines, will result in improved patient care. [Level 5]