Impact of elevated direct bilirubin levels on the measurement of unbound bilirubin

BACKGROUND: In high-risk neonates, such as very low birth weight infants or those undergoing abdominal surgery, elevated direct bilirubin (DB) levels are frequently observed. Under such conditions, unbound bilirubin (UB) measured using peroxidase-based analyzers may appear spuriously elevated, complicating clinical interpretation. METHODS: Retrospective analysis was performed on laboratory datasets with complete measurements of total bilirubin (TB), DB, UB, and albumin from January 2021 to December 2023. DB was measured enzymatically using the Nescauto VL D-bil bilirubin oxidase method. Indirect bilirubin (iDB) was calculated as TB minus DB, and its molar ratio to albumin (iDB/albumin) was evaluated for correlation with UB across varying DB levels and DB/TB ratios. Outlier-high UB values were defined as those exceeding the 95% confidence interval of the iDB/albumin ratio within the physiological range (DB < 1 mg/dL and DB/TB < 10%). RESULTS: A total of 5970 datasets from 1386 neonates were analyzed. As DB levels and DB/TB ratios increased, the correlation between the iDB/albumin ratio and UB weakened, and the regression slope became steeper. The proportion of outlier-high UB values rose significantly: 4.9%, 10.8%, 32.5%, and 92.2% for DB