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.

Figures

Figure 1

Bfeq measured by the FloPro Analyzer (2-fold sample dilution) versus Bfeq measured by the UB Analyzer (42-fold sample dilution). The correlation is significant and the regression equation, regression coefficient, and level of significance are shown.

Figure 2

Bfeq measured by the UB Analyzer as a function of the TBC. The regression equation for Bfeq versus TBC is shown and the variability in bilirubin-albumin binding in the population is reflected in the correlation coefficient of 0.41.

Figure 3

The difference between Bf-100% and Bfeq as a function of Bf-100% as measured with the UB Analyzer. The regression line, which increases significantly with increasing Bf-100%, is shown along with the regression equation and correlation coefficient.

Figure 4

The difference between Bf-100% and Bfeq as a function of Bf-100% as measured with the Global FloPro Analyzer. The regression line does not increase significantly with increasing Bf-100.

Figure 5

UB Analyzer measurements of B feq with and without Cl- in the reaction buffer. Bfeq with Cl- is plotted against B without Cl- feq. The regression equation, correlation coefficient, and level of significance are shown.