Effects of sample dilution, peroxidase concentration, and chloride ion on the measurement of unbound bilirubin in premature newborns

Objectives To assess the effects of sample dilution, peroxidase concentration, and chloride ion (Cl−) on plasma unbound bilirubin (Bf) measurements made using a commercial peroxidase methodology (UB Analyzer) in a study population of ill, premature newborns. Design and methods Bf was measured with a UB Analyzer in 74 samples at the standard 42-fold sample dilution and compared with Bf measured at a 2-fold sample dilution using a FloPro Analyzer. Bf was measured at two peroxidase concentrations to determine whether the peroxidase steady state Bf (Bfss) measurements were significantly less than the equilibrium Bf (Bfeq), in which case it was necessary to calculate Bfeq from the two Bfss measurements. Bf was also measured before and after adding 100 mmol/L Cl− to the UB Analyzer assay buffer. Results Bfeq at the 42-fold dilution was nearly 10-fold less than but it correlated significantly with Bfeq at the 2-fold dilution (mean 8.2 ± 5.2 nmol/L versus 73.5 ± 70 nmol/L, respectively, p < 0.0001; correlation r = 0.6). The two UB Analyzer Bfss measurements were significantly less than Bfeq in 42 of 74 (57%) samples, and Cl− increased Bfeq in 66 of 74 (89%) samples by a mean of 82 ± 67%. Conclusions Bfss measured by the UB Analyzer at the standard 42-fold sample dilution using assay buffer without Cl− and a single peroxidase concentration is significantly less than the Bfeq in undiluted plasma. Accurate Bf measurements can be made only in minimally diluted serum or plasma.