Effect of storage and freezing on unbound bilirubin measurement

Unbound bilirubin (Bf) 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 Bf analysis is not currently available in clinical laboratories. The samples are often stored at varying temperature and for varying duration before Bf analysis.

We evaluated the effect of storing blood samples up to 1 month at -20 degrees C and -80 degrees C on Bf measurement. In a subset of samples collected for neonates enrolled in a prospective Bf study, TSB (micromol/l) and Bf (micromol/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 Bf measured again.

There was no effect of storage for 2 weeks at either -20 degrees C or -80 degrees C on Bf measurement. There appears to be no effect on Bf after storage at -80 degrees C for a month. However, there was significant decrease in Bf after storage at -20 degrees C for a month.

The results of this study suggest that blood samples for Bf measurement should be analyzed within 2 weeks after collection and storage at -20 degrees C.