How do you calculate unconjugated (indirect) bilirubin levels?

How to Calculate Unconjugated Bilirubin

Unconjugated (indirect) bilirubin is calculated by subtracting the direct (conjugated) bilirubin from the total bilirubin level. 1

Understanding Bilirubin Types

• Bilirubin exists in two main forms: unconjugated and conjugated. Unconjugated bilirubin is insoluble and transported to the liver, where it is converted to soluble conjugated bilirubin for excretion 1
• Most laboratories routinely report total bilirubin, which comprises both unconjugated and conjugated fractions 1
• It's important to note that the terms "direct" and "conjugated" hyperbilirubinemia are often incorrectly used interchangeably. Direct bilirubin includes both the conjugated fraction and delta bilirubin (bound to albumin) 1

Calculation Method

1. Basic Formula: Unconjugated Bilirubin = Total Bilirubin - Direct Bilirubin 1

2. Laboratory Reporting:

   • Most clinical laboratories will report both total bilirubin and direct bilirubin 1
   • Some advanced laboratories may directly measure unconjugated bilirubin using specialized techniques 2

Clinical Considerations

• In patients with cholestasis, calculating unconjugated bilirubin by subtraction may lead to overestimation by up to 5-10 mg/dL 2
• For more accurate measurement in patients with cholestatic liver disease, direct measurement of unconjugated bilirubin using specialized methods (like multilayered slides or HPLC) is preferred over calculation 2, 3
• In Gilbert's syndrome, a common cause of isolated elevated bilirubin, unconjugated bilirubin should be less than 20-30% of the total bilirubin in the absence of hemolysis 1

Special Situations

• In neonates, physiological increases in total bilirubin are primarily unconjugated 1
• When evaluating hyperbilirubinemia in clinical trials for cholestatic liver diseases, diagnosis of Gilbert's Syndrome is confirmed by calculating the amount of conjugated bilirubin, which should be less than 20%-30% of the total bilirubin 1
• If the etiology of prolonged hyperbilirubinemia is uncertain, a breakdown of the direct bilirubin fraction to conjugated and delta bilirubin should be considered 1

Advanced Measurement Methods

• High-performance liquid chromatography (HPLC) is considered the most reliable method for measuring bilirubin fractions but is impractical for routine use 4
• Some specialized analyzers use direct spectrophotometry to measure unconjugated bilirubin and the sum of bilirubin conjugates 4
• Enzymatic methods can measure conjugated bilirubins at pH 10, which is more helpful for differential diagnosis of jaundice than direct bilirubin measurement 4

Common Pitfalls

• Do not subtract direct-reacting (conjugated) bilirubin from total bilirubin when using guidelines for phototherapy and exchange transfusion in neonates 1
• In cases where direct bilirubin is 50% or more of total bilirubin, standard calculation methods may not be reliable, and expert consultation is recommended 1
• The presence of delta bilirubin (bound to albumin) can cause direct hyperbilirubinemia to persist longer due to its 21-day half-life 1