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

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. 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. 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%. 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.