The laboratory method as a variable in the diagnosis of hyperbilirubinemia

Algorithms based on fractionation of bilirubin into direct-reacting and indirect-reacting fractions by diazo methods are frequently utilized for investigation of hyperbilirubinemia. Unfortunately, direct-reacting and indirect-reacting fractions do not correspond precisely with conjugated and unconjugated pigment. Advances in analytical methods allow accurate quantitation of the individual fractions of bilirubin in serum. Three cases of hyperbilirubinemia are presented in which bilirubin fractionation by diazo methods suggested an erroneous diagnosis. Analysis of the serum samples by high-performance liquid chromatography for bilirubin conjugates confirmed the correct clinical diagnosis. Diazo methods for bilirubin analysis are subject to marked discrepancies. While the new methodology utilizing high-performance liquid chromatography is not practical for routine bilirubin analysis, optimization of diazo methods utilizing rigid and meticulous protocol is suggested. Laboratories performing diazo bilirubin determinations should correlate their results with those of other laboratories and clinical data. Clinicians need to be aware of the reliability of a particular test result before embarking on a lengthy and expensive evaluation.