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

Abstract: Objectives : To assess the effects of sample dilution, peroxidase concentration, and chloride ion (Cl−) on plasma unbound bilirubin (B f) measurements made using a commercial peroxidase methodology (UB Analyzer) in a study population of ill, premature newborns. Design and methods : B f was measured with a UB Analyzer in 74 samples at the standard 42-fold sample dilution and compared with B f measured at a 2-fold sample dilution using a FloPro Analyzer. B f was measured at two peroxidase concentrations to determine whether the peroxidase steady state B f (B fss) measurements were significantly less than the equilibrium B f (B feq), in which case it was necessary to calculate B feq from the two B fss measurements. B f was also measured before and after adding 100 mmol/L Cl− to the UB Analyzer assay buffer. Results : B feq at the 42-fold dilution was nearly 10-fold less than but it correlated significantly with B feq 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 B fss measurements were significantly less than B feq in 42 of 74 (57%) samples, and Cl− increased B feq in 66 of 74 (89%) samples by a mean of 82±67%. Conclusions : B fss 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 B feq in undiluted plasma. Accurate B f measurements can be made only in minimally diluted serum or plasma. [Copyright &y& Elsevier]