Effect of storage and freezing on unbound bifirubin measurement

Background: Unbound bilirubin (B(f)) is a better predictor of bilirubin toxicity than total serum bilirubin (TSB) in the postnatal period. The Food and Drug Administration approved peroxidase test for B(f) analysis is not currently available in clinical laboratories. The samples are often stored at varying temperature and for varying duration before B(f) analysis. Methods: We evaluated the effect of storing blood samples up to 1 month at -20 degrees C and -80 degrees C on B(f) measurement. In a subset of samples collected for neonates enrolled in a prospective B(f) study, TSB (mu mol/l) and B(f) (mu mol/l) were measured by the peroxidase test and then the samples were immediately frozen at -20 degrees C (n= 10) or -80 degrees C (n= 10) for either 2 weeks or at -20 degrees C (n = 16) or -80 degrees C (n = 10) for 1 month. After the designated freezing period, the samples were thawed and TSB and B(f) measured again. Results: There was no effect of storage for 2 weeks at either -20 degrees C or -80 degrees C on B(f) measurement. There appears to be no effect on B(f) after storage at -80 degrees C for a month. However, there was significant decrease in B(f) after storage at -20 degrees C for a month. Conclusions: The results of this study suggest that blood samples for B(f) measurement should be analyzed within 2 weeks after collection and storage at -20 degrees C. (c) 2008 Elsevier B.V. All rights reserved.