Bilirubin (Jendrassik–Grof / diazo method)

Principle

• Diazo reaction: bilirubin couples with diazotised sulfanilic acid to form azobilirubin.
• Conjugated (direct) bilirubin reacts in aqueous solution without an accelerator.
• Unconjugated bilirubin is albumin-bound and reacts only when an accelerator is added; Jendrassik–Grof uses caffeine–sodium benzoate (Malloy–Evelyn uses methanol).
• In Jendrassik–Grof, ascorbic acid stops the reaction and alkaline tartrate converts the red azobilirubin to a blue form, read at about 600 nm, which reduces interference from haemoglobin and carotenoids.

Specimen

• Serum or heparin plasma, protected from light; bilirubin is photo-degraded and results fall on exposure.
• Avoid haemolysis: haemoglobin inhibits the diazo reaction (falsely low).

Procedure

• Total: sample + accelerator + diazo reagent; timed incubation; add tartrate; read.
• Direct: sample + diazo reagent without accelerator; read at a fixed time.
• Sample blanks (no diazo) correct for sample colour.
• Indirect (unconjugated) = total – direct.

Results

• Typical adult total bilirubin 0.3–1.2 mg/dL (5–21 µmol/L); direct up to about 0.3 mg/dL (≤5 µmol/L). Local reference ranges apply.
• Conversion: mg/dL × 17.1 = µmol/L.

Interpretation

• Mainly unconjugated rise: haemolysis, Gilbert syndrome, Crigler–Najjar, physiological neonatal jaundice.
• Mainly conjugated rise: biliary obstruction, hepatocellular injury, Dubin–Johnson and Rotor syndromes.
• Delta bilirubin (conjugated bilirubin covalently bound to albumin) reacts as direct and persists after obstruction resolves because of albumin's long half-life.

Quality control and pitfalls

• Neonatal samples need methods calibrated for high concentrations; results above linearity need dilution.
• Lipaemia may affect readings.

Clinical use

• Evaluation of jaundice, liver disease, haemolysis and neonatal hyperbilirubinaemia.