Direct Spectrophotometric Method for Measurement of Bilirubin in Newborns: Comparison with HPLC and an Automated Diazo Method

Abstract

The measurement of serum total bilirubin is one of the most frequently performed tests in newborns (1)(2)(3). Direct spectrophotometry for the measurement of bilirubin in sera from newborns is a simple and rapid method that requires minimal sample for analysis (4). The direct spectrophotometric assay is based on the absorbance of bilirubin at 454 nm; by contrast, hemoglobin absorbs equally at both 454 and 528 nm. Subtraction of the absorbance at 528 nm from that at 454 nm eliminates the effect of hemolysis and yields a value that can be attributed primarily to bilirubin. Unfortunately, other pigments, such as carotenoids, also absorb at 454 nm, thus limiting this technique to neonates <2–3 weeks of age. Direct spectrophotometric methods have been compared with diazo (Jendrassik–Grof-based) bilirubin methods (5)(6)(7)(8)(9), but sample hemolysis can increase or decrease the results obtained by the latter procedure, depending on the concentrations of hemoglobin and bilirubin in the sample (10)(11). Most such studies either do not describe the effect of sample hemolysis on the agreement between methods or report poorer agreement between methods or increased scatter around the regression line (6)(8)(9). HPLC for measurement of serum bilirubin concentrations is labor-intensive and not practical for routine use; …