Direct (conjugated) bilirubin reacts with the diazonium salt 2,4-dichlorophenyldiazonium (2,4-DPD) in the presence of sulfamic acid, forming azobilirubin. This colored complex can be measured photometrically at 546 nm. Of the two fractions of bilirubin present in serum, bilirubin glucuronate (conjugated) and bilirubin free associated with albumin (unconjugated), only the first reacts directly, while the free bilirubin needs to be dissociated from the protein by an accelerator to react. Indirect bilirubin is calculated by the difference between total bilirubin (with accelerator) and direct bilirubin (without accelerator). The concepts of 'direct' and 'indirect' refer exclusively to the reaction characteristics in the presence or absence of accelerators or solubilizers and are only approximate equivalents to the two mentioned bilirubin fractions.
