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1.  Influence of Clinical Status on the Association Between Plasma Total and Unbound Bilirubin and Death or Adverse Neurodevelopmental Outcomes in Extremely Low Birth Weight Infants 
Objectives
To assess the influence of clinical status on the association between total plasma bilirubin and unbound bilirubin on death or adverse neurodevelopmental outcomes at 18–22 months corrected age in extremely low birth weight infants.
Method
Total plasma biirubin and unbound biirubin were measured in 1,101 extremely low birth weight infants at 5±1 day of age. Clinical criteria were used to classify infants as clinically stable or unstable. Survivors were examined at 18–22 months corrected age by certified examiners. Outcome variables were death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss, and death prior to follow-up. For all outcomes, the interaction between bilirubin variables and clinical status was assessed in logistic regression analyses adjusted for multiple risk factors.
Results
Regardless of clinical status, an increasing level of unbound bilirubin was associated with higher rates of death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss and death before follow-up. Total plasma bilirubin values were directly associated with death or neurodevelopmental impairment, death or cerebral palsy, death or hearing loss, and death before follow-up in unstable infants, but not in stable infants. An inverse association between total plasma bilirubin and death or cerebral palsy was found in stable infants.
Conclusions
In extremely low birth weight infants, clinical status at 5 days of age affects the association between total plasma and unbound bilirubin and death or adverse neurodevelopmental outcomes at 18–22 months of corrected age. An increasing level of UB is associated a higher risk of death or adverse neurodevelopmental outcomes regardless of clinical status. Increasing levels of total plasma bilirubin are directly associated with increasing risk of death or adverse neurodevelopmental outcomes in unstable, but not in stable infants.
doi:10.1111/j.1651-2227.2010.01688.x
PMCID: PMC2875328  PMID: 20105142
Plasma bilirubin; unbound bilirubin; Extremely low birth weight infants; Neurodevelopmental outcomes
2.  Effects of Sample Dilution, Peroxidase Concentration, and Chloride Ion on the Measurement of Unbound Bilirubin in Premature Newborns 
Clinical biochemistry  2006;40(3-4):261-267.
Objectives
To assess the effects of sample dilution, peroxidase concentration, and chloride ion (Cl-) on plasma unbound bilirubin (Bf) measurements made using a commercial peroxidase methodology (UB Analyzer) in a study population of ill, premature newborns.
Design and Methods
Bf was measured with a UB Analyzer in 74 samples at the standard 42-fold sample dilution and compared with Bf measured at a 2-fold sample dilution using a FloPro Analyzer. Bf was measured at two peroxidase concentrations to determine whether the peroxidase steady state Bf (Bfss) measurements were significantly less than the equilibrium Bf (Bfeq), in which case it was necessary to calculate Bfeq from the two Bfss measurements. Bf was also measured before and after adding 100 mmol/L Cl- to the UB Analyzer assay buffer.
Results
Bfeq at the 42-fold dilution was nearly 10-fold less than but correlated significantly with Bfeq 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 Bfeq in 42 of 74 (57%) samples, and Cl- increased Bfeq in 66 of 74 (89%) samples by a mean of 82±67%.
Conclusions
Bfss 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 Bfeq in undiluted plasma. Accurate Bf measurements can be made only in minimally diluted serum or plasma.
doi:10.1016/j.clinbiochem.2006.09.006
PMCID: PMC1945224  PMID: 17069786
newborn jaundice; hyperbilirubinemia; unbound bilirubin; peroxidase test; bilirubin/albumin binding; free bilirubin

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