A 2,4-dichlorophenyl diazonium-based method for total bilirubin without interference from indican in uremic sera

Comparison of total bilirubin quantification by a 2,4-dichlorophenyl diazonium method (2,4-DCPD) with a Jendrassik-Grof type of method showed excellent correlation for randomly selected sera. However, sera from uremic patients on chronic hemodialysis showed a marked positive bias for the 2,4-DCPD result as compared with the Jendrassik-Grof result. The mean difference was 5.3 mg/L, and resulted in about 20% of the hemodialysis patients having bilirubin values greater than 13 mg/L, the upper limit of our reference range. Indican in uremic sera reportedly reacts with certain diazo reagents, so we investigated indican's reactivity in the above methods. In vitro addition of indican caused no interference in the Jendrassik-Grof method, but produced a significant positive interference in the 2,4-DCPD method, 1 mmol of indican per liter appearing as about 36 mg of total bilirubin per liter. Long reaction times with the 2,4-DCPD reagent accentuate the problem. By shortening the reaction time with the 2,4-DCPD reagent to 1.7 min, we find that the indican interference can be eliminated, without affecting quantification of total bilirubin in either normal or uremic sera.