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

• Published in: Clinical biochemistry Vol. 40; no. 3; pp. 261 - 267
• Main Authors: Ahlfors, Charles E., Vreman, Hendrik J., Wong, Ronald J., Bender, G. Jesse, Oh, William, Morris, Brenda H., Stevenson, David K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2007
• Subjects: Analytic Sample Preparation Methods; Bilirubin - blood; Bilirubin/albumin binding; Chlorides - chemistry; Diagnostic Techniques and Procedures - instrumentation; Diagnostic Techniques and Procedures - standards; Female; Free bilirubin; Humans; Hyperbilirubinemia; Infant, Newborn; Infant, Premature - blood; Jaundice, Neonatal - diagnosis; Newborn jaundice; Peroxidase test; Peroxidases - chemistry; Unbound bilirubin; B f-100; kp; B feq; Bilirubin/albumin binding; B f; Cl; K; NRN; A 460; B f-50; Peroxidase test; Hyperbilirubinemia; TBC; Unbound bilirubin; Newborn jaundice; Free bilirubin; B fss; CPMC
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/j.clinbiochem.2006.09.006

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.