Conjugated Hyperbilirubinemia Workup

Updated: Aug 31, 2021
  • Author: Heba Rashid Ashraf, MD; Chief Editor: BS Anand, MD  more...
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Approach Considerations

Appropriate initial laboratory testing in cases of suspected conjugated hyperbilirubinemia depends on the clinical history and physical examination findings. [3, 4, 18, 19, 20, 21] Misdiagnosis of cholestatic jaundice potentially delays the identification of severe liver disease; investigate all cases of prolonged neonatal jaundice (>2 weeks after birth). [22, 23]

Liver biopsy is indicated in cases with causes in which irreversible liver damage may occur, [24] such as biliary atresia, the most common cause of neonatal cholestasis. [22, 25] Talachian et al reported a significant delay between pediatric patients presenting with infantile cholestasis and subsequently undergoing liver biopsy in the setting of potential irreversible liver damage. [24]

The gold standard for diagnosing biliary atresia is intraoperative cholangiography and histologic evaluation of the duct remnant. [21]


Laboratory Studies


Obtain fractionated bilirubin levels. [21] Bilirubin can be measured in "direct" and "indirect" forms. These forms reflect the concentration of conjugated and unconjugated levels respectively.

The van den Bergh method involves the reaction of bilirubin with diazotized sulfanilic acid that forms dipyrryl azopigments and can be detected spectrophotometrically. Because of its limited aqueous solubility, unconjugated bilirubin reacts slowly in the absence of an accelerator, such as ethanol, whereas conjugated bilirubin reacts rapidly. Total bilirubin is measured in the presence of an accelerator, whereas directly reacting bilirubin is measured without an accelerator. Indirectly reacting bilirubin is calculated by subtracting the directly reacting bilirubin score from the total bilirubin score. [26] Note that although the directly reacting bilirubin concentration approximates the conjugated bilirubin concentration in most cases, the two terms do not mean the same thing. Similarly, indirect bilirubin is not the same as unconjugated bilirubin.

However, the van den Bergh method is dated and may not accurately show the concentration of conjugated and unconjugated bilirubin owing to other substances interfering with the diazo reaction. Chromatographic analysis, such as high-performance liquid chromatography (HPLC) and reflectance fluorometry is a more sensitive test for bilirubin.

Delta bilirubin is bilirubin that is covalently bound to albumin through a nonenzymatic process. This covalent bond prevents delta bilirubin from filtration, thus, it cannot be excreted through urine or bile. The serum concentration of delta bilirubin rises with prolonged cholestasis and hepatobiliary obstruction. [27]

The kidneys do not filter unconjugated bilirubin because of its avid binding to albumin. For this reason, the presence of bilirubin in the urine indicates the presence of conjugated hyperbilirubinemia.

Devgun et al indicate a direct bilirubin concentration of 10 μmol/L or higher should be used to consider the presence of conjugated hyperbilirubinemia, provided that the total bilirubin concentration is also above the reference interval. [28] The North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition consider a direct/conjugated bilirubin level above 1.0 mg/dL (17 μmol/L) as abnormal in the presence of an elevated total bilirubin. [21] In newborns, rule out infection for levels of conjugated bilirubin of 0.5 mg/dL and above, up to 2 mg/dL, and monitor the infant; for those with conjugated bilirubin levels of 2 mg/dL and higher, a more thorough evaluation of the hepatobiliary system is warranted. [26, 29]

For formula-fed infants who are jaundiced after age 2 weeks or otherwise-well, breast-fed infants who are icteric at age 3 weeks, obtain serum total and conjugated bilirubin levels. [21]

Other laboratory studies

Obtain the following laboratory studies for all patients with suspected conjugated hyperbilirubinemia:

  • Complete blood cell (CBC) count to screen for hemolysis

  • Serum aminotransferases (aspartate aminotransferase [AST], alanine aminotransferase [ALT])

  • Serologic screen for viral hepatitis, including hepatitis C virus (HCV) antibody and hepatitis B surface antigen (HBsAg) or anti-hepatitis B core antibody (anti-HBcAb)

  • Alkaline phosphatase (ALP): If elevated or if an obstruction is suspected, images of the bile ducts should be obtained. Gamma-glutamyl transpeptidase (GGTP) results may help differentiate a hepatic source of the elevated ALP from bone or other causes.

  • Blood alcohol or acetaminophen levels upon admission (may be useful in certain cases).

  • Antimitochondrial antibody when considering primary biliary cholangitis

  • Antinuclear antibodies (ANAs), smooth-muscle antibodies, and other serologic studies when considering autoimmune hepatitis

  • Iron and genetic studies when considering hemochromatosis

  • Copper studies when considering Wilson disease

  • Alpha-1 antitrypsin fractionation and other studies when considering hereditary liver diseases


Imaging Studies


Abdominal ultrasonography should be performed to exclude biliary obstruction and to evaluate the liver parenchyma for possible cirrhosis, tumor, steatosis, or congestion. [21]

Ultrasonography is safe, noninvasive, and portable. This image modality provides good visualization of the gallbladder, bile ducts, and cystic lesions, and it can detect parenchymal liver disease, such as cirrhosis or infiltration, and signs of portal hypertension.

However, ultrasonography also has a limited resolution, and it may not detect common bile duct stones because of the presence of bowel gas.

Computed tomography (CT) scanning

Abdominal CT scans provide additional information about patients with abnormal ultrasonography scans. CT scanning may be the initial imaging modality in some cases.


CT scanning offers the following advantages:

  • Better resolution than ultrasonography

  • Provides good evaluation of the entire bile duct

  • Can define the anatomy better than ultrasonography, especially if contrast agents are used

  • Better for evaluating suspected malignancies, especially with evaluation of the arterial phase

  • Permits guided needle biopsies


Disadvantages of CT scanning include the following:

  • More expensive and less portable than ultrasonography

  • Results in radiation exposure

  • Requires intravenous (IV) contrast medium for best results

  • Less sensitive than ultrasonography for gallbladder stones

Magnetic resonance imaging (MRI)

Abdominal MRI produces images comparable in quality to CT scans without patient exposure to ionizing radiation. Following the administration of suitable contrast agents, detailed imaging of the biliary tract is possible. MR cholangiopancreatography (MRCP) may be particularly useful when evaluating cholestasis of pregnancy or patients who are too debilitated to tolerate traditional cholangiography.


MRI offers the following advantages:

  • Requires no exposure to ionizing radiation (ie, safe in pregnancy)

  • Permits use of multiple contrast agents and multiple scanning techniques, which enhance potential information content

  • Permits guided needle biopsies (open MRI systems only)

  • Ability to evaluate bile and pancreatic ducts with special contrast agents


Disadvantages of MRI include the following:

  • Not universally available

  • Cannot be used in most patients with metallic implants

  • Requires IV contrast medium for best results

  • Clinical experience is still somewhat limited



Endoscopic retrograde cholangiopancreatography (ERCP)

ERCP is useful in cases where biliary obstruction is strongly suspected. It is the investigation of choice to detect and treat common bile duct stones and is also useful for making a diagnosis of pancreatic cancer. Other conditions in which ERCP may be useful include primary sclerosing cholangitis and the presence of choledochal cysts.


ERCP offers the following advantages:

  • Allows treatment of obstruction using sphincterotomy, stone extraction, stent placement, or balloon-dilation of strictures

  • Permits biopsies under direct visualization

  • Provides excellent visualization of the bile ducts


Disadvantages of ERCP include the following:

  • Requires conscious sedation and results in radiation exposure

  • May cause pancreatitis, perforation, bleeding, and other complications that may result in death

  • Not always successful if not performed in specialized center with expertise, especially after gastroduodenal surgery

Percutaneous transhepatic cholangiography (PTC or PTHC)

PTC or PTHC offers most of the diagnostic and therapeutic possibilities of ERCP and may be more readily available in some settings. It can be useful when ERCP has been unsuccessful or is not available.


PTC or PTHC offers the following advantages:

  • Successful in most cases of biliary obstruction

  • Allows treatment of obstruction by stone extraction, balloon-dilation of strictures, or stent placement

  • Permits biopsies or brush cytology

  • Provides excellent visualization of the bile ducts


Disadvantages of PTC or PTHC include the following:

  • Typically more invasive than ERCP

  • May not be successful unless the bile ducts are dilated

  • Results in radiation exposure and requires the use of contrast medium