The Effect of Conjugated Bilirubin on the Measurement of Unbound Bilirubin

Unbound bilirubin (Bf) is a stronger predictor of neurotoxicity in newborns than that of total serum bilirubin (TSB). Standard Bf assays rely on peroxidase methods (e.g., Arrows analyser), whereas UBCheck employs a near-infrared fluorescence sensor. Because conjugated bilirubin (CBR) levels are often elevated in infants, they may alter Bf measurement accuracy, particularly with peroxidase-based methods. This study compared CBR effects on Bf measurement results from the UBCheck and Arrows assays and evaluated whether they reflected albumin displacement, assay interference, or both.

Bilirubin-albumin complexes were spiked with CBR (0.1-5 mg/dL). Bf was measured with both assays using a three-stage paired-sample protocol to assess displacement and interference.

At CBR ≤ 0.2 mg/dL, both assays yielded comparable Bf values. At 0.5-3 mg/dL, Arrows showed 12%-120% Bf increases, while UBCheck remained stable. At 4 mg/dL, UBCheck rose 20%, and Arrows increased by 220%. High CBR acted as both interferent and displacer.

In the presence of neonatal CBR concentrations, UBCheck provides reliable Bf measurements, whereas Arrows substantially overestimates Bf, limiting its clinical utility.