How to Fractionate Bilirubin
Order a laboratory test that measures total bilirubin and direct (conjugated) bilirubin, then calculate indirect (unconjugated) bilirubin by subtracting direct from total bilirubin. 1
Standard Clinical Approach
The fractionation of bilirubin in routine clinical practice involves:
- Order total bilirubin and direct bilirubin through standard laboratory testing using the diazo reaction method, which is the current gold-standard and most widely available 2
- Calculate indirect (unconjugated) bilirubin by subtracting direct bilirubin from total bilirubin 3
- Interpret the predominant fraction to guide differential diagnosis: unconjugated elevation suggests hemolysis or impaired conjugation (Gilbert's syndrome), while conjugated elevation indicates hepatobiliary disease 3
Key Clinical Considerations
When to Fractionate
Fractionate total bilirubin whenever it is elevated to determine the percentage derived from direct (conjugated) bilirubin, as recommended by consensus guidelines for cholestatic liver disease 1
Understanding Direct vs. Conjugated Bilirubin
A critical pitfall exists in terminology:
- Direct bilirubin includes both conjugated bilirubin AND delta bilirubin (albumin-bound), which has a half-life of approximately 21 days and causes prolonged hyperbilirubinemia 1
- The terms "direct" and "conjugated" are incorrectly used interchangeably in clinical practice 1
- If prolonged hyperbilirubinemia is unexplained, consider requesting breakdown of direct bilirubin into conjugated and delta fractions 1
Diagnostic Thresholds
- Conjugated (direct) bilirubin >35% of total typically indicates drug-induced liver injury (DILI) or hepatobiliary disease 1
- Unconjugated bilirubin <20-30% of total in the absence of hemolysis confirms Gilbert's syndrome 1
- In neonates, conjugated bilirubin >25 μmol/L requires urgent pediatric referral for possible liver disease 3
Advanced Fractionation Methods
High-Performance Liquid Chromatography (HPLC)
HPLC is the gold-standard method for complete bilirubin fractionation but is impractical for routine clinical use 4, 5
HPLC separates bilirubin into five distinct fractions:
- Delta bilirubin (albumin-bound) 5, 6
- Bilirubin diglucuronide (BDG) 5, 6
- Bilirubin monoglucuronide (BMG) 5, 6
- Photoisomers 5
- Unconjugated bilirubin 5, 6
When HPLC May Be Indicated
- Research settings or complex diagnostic dilemmas where standard fractionation is insufficient 4, 5
- Monitoring phototherapy in neonates to quantify photoderivatives 5
- Following jaundice progression using ratios like B delta/(B delta + BDG + BMG) in hepatobiliary disease 6
Common Pitfalls to Avoid
Laboratory Method Variability
Different laboratories may yield conflicting results, particularly when total bilirubin is <5 mg/dL 7
- Marked variations exist among laboratories in their ability to reliably fractionate sera with mild hyperbilirubinemia 7
- Confirm your laboratory's reliability for low-level bilirubin fractionation, especially when differentiating Gilbert's syndrome from early hepatobiliary disease 7
Misdiagnosis of Gilbert's Syndrome
- Gilbert's syndrome causes unconjugated hyperbilirubinemia with reduced glucuronyltransferase activity to 20-30% of normal 1, 3
- Total bilirubin rarely exceeds 4-5 mg/dL in Gilbert's syndrome 1
- Confirm diagnosis by calculating conjugated bilirubin <20-30% of total in the absence of hemolysis 1
- Consider genetic testing for DNA mutations of UDP-glucuronosyltransferase if diagnosis remains uncertain 1
Isolated Direct Hyperbilirubinemia
Monitor any persistent isolated elevations in direct bilirubin closely, especially in patients with underlying cholestatic liver disease, as this may indicate DILI even without aminotransferase elevation 1