Liver Function Test PDF

## WK16 LEC Liver Function Test ### Introduction The liver is a very large and complex organ responsible for performing vital tasks that impact all body systems. Its complex functions include: * metabolism of carbohydrates, lipids, proteins, and bilirubin * detoxification of harmful substances...

## WK16 LEC Liver Function Test ### Introduction The liver is a very large and complex organ responsible for performing vital tasks that impact all body systems. Its complex functions include: * metabolism of carbohydrates, lipids, proteins, and bilirubin * detoxification of harmful substances * storage of essential compounds * excretion of substances to prevent harm The liver is a relatively resilient organ that can regenerate cells that have been destroyed by some short-term injury or disease. However, if the liver is damaged repeatedly over a long period, it may undergo irreversible changes that permanently interfere with its essential functions. ### Gross Anatomy * Right Lobe of Liver: Approximately six times larger than the left lobe. * Left Lobe of Liver * Falciform Ligament: Divides unequally the liver into two lobes. * Hepatocyte * Kupffer cells (phagocytic cells in liver) ### Two Major Sources of Blood Supply * **Hepatic Artery:** A branch of the aorta that supplies oxygen-rich blood from the heart to the liver. * **Portal Vein:** Supplies nutrient-rich blood (collected as food is digested) from the digestive tract. ### Excretory system of the liver * The excretory system of the liver begins at the **bile canaliculi**, small spaces between the hepatocytes that form intrahepatic ducts. * The intrahepatic ducts join to form the **right and left hepatic ducts**, which drain the secretions from the liver. * The right and left hepatic ducts merge to form the **common hepatic duct**, which is eventually joined with the cystic duct of the gallbladder to form the **common bile duct**. * Combined digestive secretions are then expelled into the duodenum. ### Microscopic Anatomy * The liver is divided into microscopic units called lobules. * Each lobule is a six-sided structure with a **centrally located vein** (called the central vein) with **portal triads** at each of the corners. * The liver contains two major cell types: * **hepatocytes** * **Kupffer cells**. ### Biochemical Functions The liver is in charge of: * Excretory & Secretory * Metabolism * Detoxification, and * Storage ### Excretory and Secretory One of the most important functions of the liver is the processing and excretion of endogenous and exogenous substances into the bile or urine such as the major heme waste product, bilirubin. * The liver is the only organ that has the capacity to rid the body of heme waste products. * Hemoglobin is degraded into heme, globin, and iron. * Bilirubin is bound by albumin and transported to the liver. * Ligandin, which is located in the hepatocyte, is responsible for transporting unconjugated bilirubin to the endoplasmic reticulum, where it may be rapidly conjugated. * The heme portion of hemoglobin is converted to bilirubin in 2 to 3 hours. * Once at the liver cell, unconjugated bilirubin flows into the sinusoidal spaces and is released from albumin so it can be picked up by a carrier protein called ligandin, which combines with secretions from the gallbladder through the cystic duct and is expelled through the common bile duct to the intestines. * Approximately 200 to 300 mg of bilirubin is produced per day, and it takes a normally functioning liver to process the bilirubin and eliminate it from the body. * The healthy adult has very low levels of **total bilirubin** (0.2 to 1.0 mg/dL) in the serum. The majority of this amount is in the **unconjugated form**. ### Metabolism * The metabolism of carbohydrates is one of the most important functions of the liver. * When carbohydrates are ingested and absorbed, the liver can do three things: * Use the glucose for its own cellular energy requirements. * Circulate the glucose for use at the peripheral tissues * Store glucose as glycogen (principal storage form of glucose) within the liver itself or within other tissues. * Lipids are metabolized in the liver during normal circumstances when nutrition is adequate and the demand for glucose is being met. * The liver is responsible for metabolizing both lipids and the lipoproteins and is responsible for gathering free fatty acids from the diet, and those produced by the liver itself, and breaking them down to produce acetyl-CoA. * Almost all proteins are synthesized by the liver except for the immunoglobulins and adult hemoglobin. * One of the most important proteins synthesized by the liver is **albumin**, which carries with it a wide range of important functions. * The liver is also responsible for synthesizing the positive and negative acute-phase reactants and coagulation proteins, and it also serves to store a pool of amino acids through protein degradation. ### Detoxification and Drug Metabolism * Every substance that is absorbed in the gastrointestinal tract must first pass through the liver; this is referred to as **first pass**. * This is an important function of the liver because it can allow important substances to reach the systemic circulation and prevents toxic or harmful substances from reaching systemic circulation. * The body has two mechanisms for detoxification of foreign materials (drugs and poisons) and metabolic products (bilirubin and ammonia). * It may bind the material reversibly to inactivate the compound. * It may chemically modify the compound so it can be excreted in its chemically modified form. ### Jaundice * The word jaundice comes from the French word jaune, which means "yellow." * It is one of the oldest known pathologic conditions reported, having been described by Hippocratic physicians. * **Jaundice** (or **icterus**) is used to describe the yellow discoloration of the skin, eyes, and mucous membranes. * It results from the retention of bilirubin, and it may also occur due to the retention of other substances. * **Hyperbilirubinemia:** Increased levels of bilirubin. ### Classification of Jaundice * **Prehepatic Jaundice:** The problem causing the jaundice occurs prior to liver metabolism. * **Hepatic Jaundice:** The problem causing the jaundice occurs when the primary problem resides in the liver. * **Posthepatic Jaundice:** Results from biliary obstructive disease that prevents the flow of conjugated bilirubin into the bile canaliculi. ### Prehepatic Jaundice * Prehepatic jaundice occurs when the problem causing the jaundice occurs prior to liver metabolism. * It is most commonly caused by an increased amount of bilirubin being presented to the liver, such as that seen in acute and chronic hemolytic anemias. * This type of jaundice may also be referred to as **unconjugated hyperbilirubinemia**, because the fraction of bilirubin increased in people with prehepatic jaundice is the unconjugated fraction. ### Hepatic Jaunice * Hepatic Jaundice occurs when the primary problem causing the jaundice resides in the liver (intrinsic liver defect or disease). * The intrinsic liver defect or disease can be due to disorders of bilirubin metabolism and transport defects (Crigler-Najjar syndrome, Dubin-Johnson syndrome, Gilbert's disease, and neonatal physiologic jaundice of the newborn) or due to diseases resulting in hepatocellular injury or destruction. * **Hepato cellular combined:** * Elevated esther direct or indirect bilirubin * Hepatic injuries caused by alcohol, parasites, etc * **Characteristics:** * Impaired cell uptake * Conjugation deficit * impaired bilirubin excretion ### Gilbert's Syndrome * Gilbert's syndrome, first described in the early twentieth century, is a benign autosomal recessive hereditary disorder that affects approximately 5% of the US population. * Gilbert's syndrome results from a genetic mutation in the UGT1A gene that produces the enzyme uridine diphosphate glucuronosyltransferase, one of the enzymes important for bilirubin metabolism. * It is characterized by **intermittent unconjugated hyperbilirubinemia**, underlying liver disease due to a defective conjugation system in the absence of hemolysis. * The hyperbilirubinemia usually manifests **during adolescence or early adulthood**. * **Total serum bilirubin** usually fluctuates between 1.5 and 3.0 mg/dL, and it rarely exceeds 4.5 mg/dL. * The UGT1A1 promoter contains the sequence (TA)6TAA. The insertion of an extra TA in the sequence, as seen in Gilbert's syndrome, reduces the expression of the UGT1A1 gene to 20% to 30% of normal values. * Gilbert's syndrome is the most common cause of jaundice, and interestingly, carries no morbidity or mortality in the majority of those affected. * It generally has no clinical consequences. ### Crigler-Najjar Syndrome * Crigler-Najjar syndrome was first described by Crigler and Najjar in 1952 as a syndrome of **chronic nonhemolytic unconjugated hyperbilirubinemia**. * Crigler-Najjar syndrome may be divided into two types: * **Type 1:** There is a complete absence of enzymatic bilirubin conjugation. * **Type II:** There is a mutation causing a severe deficiency of the enzyme responsible for bilirubin conjugation. ### Dubin-Johnson Syndrome * Dubin-Johnson syndrome is a rare autosomal recessive inherited disorder caused by a deficiency of the canalicular multidrug resistance/multispecific organic anionic transporter protein (MDR2/cMOAT). * This results in accumulation of conjugated and, to some extent, unconjugated bilirubin in the blood, leading to **hyperbilirubinemia** and **bilirubinuria**. * Dubin-Johnson is a condition that is obstructive in nature, so much of the conjugated bilirubin circulates bound to albumin. This type of bilirubin (conjugated bilirubin bound to albumin) is referred to as delta bilirubin. * A distinguishing feature of Dubin-Johnson syndrome is the appearance of **dark-stained granules** on a liver biopsy sample. * Usually, the **total bilirubin concentration** remains between 2 and 5 mg/dL, with more than 50% due to the conjugated fraction. * This syndrome is relatively **mild in nature** with an excellent prognosis. People with Dubin-Johnson syndrome have a **normal life expectancy**. * No treatment is necessary. ### Rotor's Syndrome * Rotor's syndrome is clinically similar to Dubin-Johnson syndrome, but the defect causing Rotor's syndrome is not known. * It is hypothesized to be due to a reduction in the concentration or activity of intracellular binding proteins such as ligandin. * Rotor's syndrome is seen less commonly than Dubin-Johnson syndrome. It carries an excellent prognosis, and therefore, treatment is not warranted. ### Physiologic Jaundice of the Newborn * Physiologic jaundice of the newborn is a result of a deficiency in the enzyme UDPGT, one of the last liver functions to be activated in prenatal life since bilirubin processing is handled by the mother of the fetus. * Premature infants may be born without UDPGT. * This deficiency results in the **rapid buildup of unconjugated bilirubin**, which can be life threatening. * When unconjugated bilirubin builds up in the neonate, it cannot be processed and is deposited in the nuclei of brain and degenerate nerve cells, causing **kernicterus**. * Infants with this type of jaundice are usually treated with **phototherapy** to destroy the bilirubin as it passes through the capillaries of the skin. * In extreme cases of hyperbilirubinemia, an **exchange transfusion** may be used as the second-line treatment when phototherapy fails. * An exchange transfusion involves removing aliquots of blood and replacing it with donor blood in order to remove abnormal blood components and circulating toxins while maintaining adequate circulating blood volume. * Because hyperbilirubinemia is so serious in newborns, bilirubin levels are carefully and frequently monitored so the dangerously high levels of unconjugated bilirubin (approximately 20 mg/dL) can be detected and treated appropriately. ### Posthepatic Jaundice * Posthepatic jaundice results from **biliary obstructive disease**, usually from physical obstructions (gallstones or tumors) that prevent the flow of conjugated bilirubin into the bile canaliculi. * Since the liver cell itself is functioning, bilirubin is effectively conjugated; however, it is unable to be properly excreted from the liver. * Since bile is not being brought to the intestines, stool loses its source of normal pigmentation and becomes clay-colored. ### Changes in Concentration of Bilirubin in Those with Jaundice | Type of Jaundice | Total Bilirubin | Conjugated Bilirubin | Unconjugated Bilirubin | |---|---|---|---| | Prehepatic | Increase | Within | Increase | | Hepatic | | | | | Gilbert's Disease | Increase | Within | Increase | | Crigler-Najjar Syndrome | Increase | Decrease | Increase | | Dubin-Johnson | Increase | Increase | Within | | Rotor's Syndrome | Increase | Increase | Within | | Jaundice of Newborn | Increase | Within | Increase | | Posthepatic | Increase | Increase | Increase | ### Cirrhosis * Cirrhosis is a clinical condition in which scar tissue replaces normal, healthy liver tissue. * As the scar tissue replaces the normal liver tissue, it blocks the flow of blood through the organ and prevents the liver from functioning properly. * Cirrhosis rarely causes signs and symptoms in its early stages, but as liver function deteriorates, the signs and symptoms appear, including fatigue, nausea, unintended weight loss, jaundice, bleeding from the gastrointestinal tract, intense itching, and swelling in the legs and abdomen. * Although some patients with cirrhosis may have prolonged survival, they generally have a poor prognosis. * Cirrhosis was the twelfth leading cause of death by disease in 2013, killing just over 36,400 people. * In the United States, the most common cause of cirrhosis is **chronic alcoholism**. * Other causes of cirrhosis include chronic hepatitis B (HBV), C (HCV), and D virus (HDV) infection, autoimmune hepatitis, inherited disorders (e.g., a1-antitrypsin deficiency, Wilson's disease, hemochromatosis, and galactosemia), nonalcoholic steatohepatitis, blocked bile ducts, drugs, toxins, and infections. * Liver damage from cirrhosis **cannot easily be reversed**, but treatment can stop or delay further progression of the disorder. * Treatment depends on the cause of cirrhosis and any complications a person is experiencing. ### Tumors * Cancers of the liver are classified as **primary or metastatic**. * **Primary liver cancer** is cancer that begins in the liver cells. * **Metastatic cancer** occurs when tumors from other parts of the body spread (metastasize) to the liver. * Tumors of the liver may also be classified as benign or malignant. * The common benign tumors of the liver include **hepatocellular adenoma** (a condition occurring almost exclusively in females of childbearing age) and **hemangiomas** (masses of blood vessels with no known etiology). * Malignant tumors of the liver include **hepatocellular carcinoma (HCC)** (also known as hepatocarcinoma, and hepatoma) and **bile duct carcinoma**. * **Hepatoblastoma** is an uncommon hepatic malignancy of children. * HCC has become increasingly important in the United States. Approximately 85% of the new cases of this liver cancer occur in developing countries, with the highest incidence of HCC reported in regions where HBV is endemic such as Southeast Asia and sub-Saharan Africa. * Approximately 80% of cases worldwide are attributable to HBV and HCV; however, the mechanism by which the infection leads to HCC is not well established. * **Whether primary or metastatic, any malignant tumor in the liver is a serious finding and carries a poor prognosis, with survival times measured in months.** ### Reye's Syndrome * Reye's syndrome is a term used to describe a group of disorders caused by infectious, metabolic, toxic, or drug-induced disease found almost exclusively in children. * Although the precise cause of Reye's syndrome is unknown, it is often preceded by a viral syndrome such as varicella, gastroenteritis, or an upper respiratory tract infection such as influenza. * Reye's syndrome is an acute illness characterized by noninflammatory encephalopathy and fatty degeneration of the liver, with a clinical presentation of profuse vomiting accompanied with varying degrees of neurologic impairment such as fluctuating personality changes and deterioration in consciousness. * The **encephalopathy** is characterized by a progression from mild confusion (stage 1) through progressive loss of neurologic function to loss of brain stem reflexes (stage 5). * The **degeneration of the liver** is characterized by a mild hyperbilirubinemia and threefold increases in ammonia and the aminotransferases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]). * Without treatment, rapid clinical deterioration leading to death may occur ### Drug- & Alcohol-Related Disorders * Drug-induced liver disease is a major problem in the United States, accounting for one-third to one-half of all reported cases of acute liver failure. * The liver is a primary target organ for adverse drug reactions because it plays a central role in drug metabolism. * Many drugs are known to cause liver damage, ranging from very mild transient forms to fulminant liver failure. Drugs can cause liver injury by a variety of mechanisms, but the most common mechanism of toxicity is via an immune-mediated injury to the hepatocytes. * Of all the drugs associated with hepatic toxicity, the most important is ethanol. In very small amounts, ethanol causes very mild, transient, and unnoticed injury to the liver; however, with heavier and prolonged consumption, it can lead to alcoholic cirrhosis. * While the exact amount of alcohol needed to cause cirrhosis is unknown, a small minority of people with alcoholism develop this condition. * Approximately 90% of the alcohol absorbed from the stomach and small intestines is transported to the liver for metabolism. * Within the liver, the elimination of alcohol requires the enzymes alcohol dehydrogenase and acetaldehyde dehydrogenase to convert alcohol to acetaldehyde and subsequently to acetate. * The acetate can then be oxidized to water and carbon dioxide, or it may enter the citric acid cycle. * Long-term excessive consumption of alcohol can result in a spectrum of liver abnormalities that may range from alcoholic fatty liver with inflammation (steatohepatitis) to scar tissue formation, as in hepatic fibrosis, to the destruction of normal liver structure seen in hepatic cirrhosis. * Alcohol-induced liver injury may be categorized into three stages: * Alcoholic fatty liver * Alcoholic hepatitis * Alcoholic cirrhosis. * The risk for the development of cirrhosis increases proportionally with the consumption of more than 30 g (the equivalent of 3 to 4 drinks) of alcohol per day, with the highest degree of risk seen with the consumption of greater than 120 g (the equivalent of 12 to 16 drinks) per day. * Alcoholic fatty liver represents the mildest category where very few changes in liver function are measurable. * This stage is characterized by slight elevations in AST, ALT, and y-glutamyltransferase (GGT), and on biopsy, fatty infiltrates are noted in the vacuoles of the liver. * This stage tends to affect young to middle aged people with a history of moderate alcohol consumption. A complete recovery within 1 month is seen when the drug is removed. * Alcoholic hepatitis presents with common signs and symptoms including fever, ascites, proximal muscle loss, and far more laboratory evidence of liver damage such as moderately elevated AST, ALT, GGT, and alkaline phosphatase (ALP) and elevations in total bilirubin greater than 5 mg/dL. * Prognosis is dependent on the type and severity of damage to the liver, and when serum creatinine levels begin to increase, it is a threatening sign, which may precede the onset of hepatorenal syndrome and death. * The last and most severe stage is **alcoholic cirrhosis**. The symptoms tend to be nonspecific and include weight loss, weakness, hepatomegaly, splenomegaly, jaundice, ascites, fever, malnutrition, and edema. * Laboratory abnormalities include increased liver function tests (AST, ALT, GGT, ALP, and total bilirubin), decreased albumin, and a prolonged prothrombin time. * A liver biopsy is the only method by which a definitive diagnosis may be made. ### Analysis of Bilirubin: Brief Review - **1883: Ehrlich** * First described the reaction of bilirubin with a diazotized sulfanilic acid solution to form a colored product using urine samples. - **1913: Van den Bergh** * Found that the diazo reaction may be applied to serum samples but only in the presence of an accelerator (solubilizer). - **1937: Malloy and Evelyn** * Developed the first clinically useful methodology for the quantitation of bilirubin in serum samples using the classic diazo reaction with a 50% methanol solution as an accelerator. - **1938: Jendrassik and Grof** * Described a method using the diazo reaction with caffeine benzoate-acetate as an accelerator. ### Bilirubinometry * Bilirubin has also been quantified using bilirubinometry in the neonatal population. * This methodology is only useful in the neonatal population because of the presence of carotenoid compounds in adult serum that causes strong positive interference in the adult population. * Bilirubinometry involves the measurement of reflected light from the skin using two wavelengths that provide a numerical index based on spectral reflectance. ### Analysis of Bilirubin * When using the several methods described earlier, two the common methods for analysis of bilirubin were identified: conjugated (direct) and unconjugated (indirect) bilirubin. * **Unconjugated (indirect) bilirubin** is a nonpolar and water-insoluble substance that is found in plasma bound to albumin. * Because of these characteristics, unconjugated bilirubin will only react with the diazotized sulfanilic acid solution (diazo reagent) in the presence of an accelerator (solubilizer). * **Conjugated (direct) bilirubin** is a polar and water-soluble compound that is found in plasma in the free state (not bound to any protein). * This type of bilirubin will react with the diazotized sulfanilic acid solution directly (without an accelerator). * **The third fraction of bilirubin is referred to as "delta" bilirubin**. * Delta bilirubin is conjugated bilirubin bound to albumin. * This fraction of bilirubin is seen only when there is significant hepatic obstruction. * Because the molecule is attached to albumin, it is too large to be filtered by the glomerulus and excreted in the urine. This fraction of bilirubin, when present, will react in most laboratory methods as conjugated bilirubin. * **Total Bilirubin = Direct Bilirubin + Indirect Bilirubin + Delta Bilirubin** * Thus, conjugated and unconjugated bilirubin fractions have historically been differentiated by solubility of the fractions. * While for many years bilirubin results were reported as direct and indirect, this terminology is now outdated. * Direct and indirect bilirubin results should be reported as conjugated and unconjugated, respectively. ### Methods to Measure Different Fractions of Bilirubin * **Bilirubin + Diazotized Sulfanilic Acid + Accelerator -**> 2 Azobilirubin (Total Bilirubin) * **Bilirubin + Diazotized Sulfanilic Acid -**> 2 Azobilirubin (Conjugated Bilirubin) * **Total Bilirubin - Conjugated Bilirubin = Unconjugated Bilirubin (Indirect Bilirubin)** ### Specimen Collection and Storage * Total bilirubin methods using a diazotized sulfanilic acid solution may be performed on either serum or plasma. * Serum, however, is preferred for the Malloy-Evelyn procedure because the addition of the alcohol in the analysis can precipitate proteins and cause interference with the method. * A fasting sample is preferred as the presence of lipemia will increase measured bilirubin concentrations. * Hemolyzed samples should be avoided as they may decrease the reaction of bilirubin with the diazo reagent. * Bilirubin is very sensitive to and is destroyed by light; therefore, specimens should be protected from light. * If serum or plasma is separated from the cells and stored in the dark, it is stable for 2 days at room temperature, 1 week at 4°C, and indefinitely at -20°C ### Methods * There is no preferred reference method or standardization of bilirubin analysis; however, the American Association for Clinical Chemistry and the National Bureau of Standards have published a candidate reference method for total bilirubin, a modified Jendrassik-Grof procedure using caffeine-benzoate as a solubilizer. * Because they both have acceptable precision and are adapted to many automated instruments, the Jendrassik-Grof or Malloy-Evelyn procedure is the most frequently used method to measure bilirubin. * The Jendrassik-Grof method is slightly more complex, but it has the following advantages over the Malloy-Evelyn method: * Not affected by pH changes * Insensitive to a 50-fold variation in protein concentration of the sample * Maintains optical sensitivity even at low bilirubin concentrations * Has minimal turbidity and a relatively constant serum blank * Is not affected by hemoglobin up to 750 mg/dL ### Malloy-Evelyn Procedure * Bilirubin pigments in serum or plasma are reacted with a diazo reagent. * The diazotized sulfanilic acid reacts at the central methylene carbon of bilirubin to split the molecule forming two molecules of azobilirubin. * This method is typically performed at pH 1.2 where the azobilirubin produced is red-purple in color with a maximal absorption of 560 nm. * The most commonly used accelerator to solubilize unconjugated bilirubin is methanol, although other chemicals have been used. ### Jendrassik-Grof Method for Total and Conjugated Bilirubin Determination * Bilirubin pigments in serum or plasma are reacted with a diazo reagent (sulfanilic acid in hydrochloric acid and sodium nitrite), resulting in the production of the purple product azobilirubin. * The product azobilirubin may be measured spectrophotometrically. * The individual fractions of bilirubin are determined by taking two aliquots of sample and reacting one aliquot with the diazo reagent only and the other aliquot with the diazo reagent and an accelerator (caffeine-benzoate). * The addition of caffeine-benzoate will solubilize the water-insoluble fraction of bilirubin and will yield a total bilirubin value (all fractions). * The reaction without the accelerator will yield conjugated bilirubin only. * After a short period the reaction of the aliquots with the diazo reagent is stopped. * The ascorbic acid destroys the excess diazo reagent. * The solution is then alkalinized using an alkaline tartrate solution, which shifts the absorbance spectrum of the azobilirubin to a more intense blue color that is less subject to interfering substances in the sample. * The final blue product is measured at 600 nm, with the intensity of color produced directly proportional to bilirubin concentration (intense color -- increased bilirubin levels). * Indirect (unconjugated) bilirubin may be calculated by subtracting the conjugated bilirubin concentration from the total bilirubin concentration. ### Reference Ranges for Bilirubin | Population | Type of Bilirubin | Reference Range | |---|---|---| | Adults | Conjugated Bilirubin | 0.0-0.2 mg/dL (0-3umol/L) | | Adults | Unconjugated Bilirubin | 0.2-0.8 mg/dL (3-14umol/L) | | Adults | Total Bilirubin | 0.2-1.0 mg/dL (3-17 umol/L) | | Premature Infants | Total Bilirubin at 24 hours |1-6 mg/dL (17-103 umol/L) | | Premature Infants | Total Bilirubin at 48 hours | 6-8 mg/dL (103-137 umol/L) | | Full-Term Infants | Total Bilirubin at 24 hours | 2-6 mg/dL (34-103 umol/L) | | Full-Term Infants | Total Bilirubin at 48 hours | 6-7 mg/dL (103-120 umol/L) | | Full-Term Infants | Total Bilirubin 3-5 days | 4-6 mg/dL (68-103 umol/L) | ### Comments and Sources of Error * Instruments should be frequently standardized to maintain reliable bilirubin results, and careful preparation of bilirubin standards is critical as these are subject to deterioration from exposure to light. * Hemolysis and lipemia should be avoided as they will alter bilirubin concentrations. * Serious loss of bilirubin occurs after exposure to fluorescent and indirect and direct sunlight; therefore, it is imperative that exposure of samples and standards to light be kept to a minimum. * Specimens and standards should be refrigerated in the dark until testing can be performed.