title: Effects of sample dilution, peroxidase concentration, and chloride ion on the measurement of unbound bilirubin in premature newborns.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: PO Box 2904, Vashon, WA 98070, USA.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Ahlfors
authors.forename: Hendrik J
authors.initials: HJ
authors.lastname: Vreman
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Wong
authors.forename: G Jesse
authors.initials: GJ
authors.lastname: Bender
authors.forename: William
authors.initials: W
authors.lastname: Oh
authors.forename: Brenda H
authors.initials: BH
authors.lastname: Morris
authors.forename: David K
authors.initials: DK
authors.lastname: Stevenson
mesh_terms: D053000:Analytic Sample Preparation Methods; D001663:Bilirubin / Q000097:blood*; D002712:Chlorides / Q000737:chemistry; D019937:Diagnostic Techniques and Procedures / Q000295:instrumentation* / Q000592:standards*; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D007234:Infant, Premature / Q000097:blood*; D007567:Jaundice, Neonatal / Q000175:diagnosis*; D010544:Peroxidases / Q000737:chemistry
chemical_list: D002712:Chlorides; D010544:Peroxidases; D001663:Bilirubin