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 (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 Bf (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 Bfss measurements were significantly less than B-feq in 42 of 74 (57%) samples, and CI- increased B-feq in 66 of 74 (89%) samples by a mean of 82 +/- 67%.