The analysis of bilirubin in serum

The most widely used methods for the assay of total bilirubin in serum are based on a coupling reaction with various diazo dyes in the presence of an accelerating agent. The 'direct' reaction, without accelerator, gives a good estimate of the conjugated and protein bound (delta) species of bilirubin only if carefully standardized reaction conditions are used. Enzymatic methods based on bilirubin oxidase appear to give similar results, for both total and direct bilirubin, and through difficult to calibrate, spectrophotometric methods can also give reliable results for total bilirubin at the higher concentrations seen in neonates. More sophisticated HPLC or dry-slide techniques are required for the specific assay of the various conjugated and protein-bound species of bilirubin in serum.