Effect of storage time, temperature and repeated freezing and thawing on serum total bilirubin

Abstract

Inadvertent delay and reanalysis may be required in bilirubin measurement due to heavy workload, transport and inadequate working staff. Hence samples have to be stored and the bilirubin concentration may need to be determined in serum samples which have to be repeatedly frozen and thawed. The objective of this study was to evaluate the effect of storage time, temperature and repeated freeze-thaw cycles on serum total bilirubin level in pooled normal serum. Bilirubin was measured by diazo-sulphanilic acid method. Following baseline measurement, serum was divided and stored in dark at 25°C and 4°C for 1, 2, 4, 6 and 8 hours respectively. Aliquots and a tube to evaluate multiple freeze-thaw cycles were stored without light exposure at -20°C for 1, 2, 3, 4, and 5 days. The total bilirubin level in serum samples stored at 25°C, 4°C and -20°C at different time intervals, did not show significant difference in mean bilirubin concentration when compared with the baseline value (p>0.05). However, the bilirubin concentration in serum sample subjected to 2nd, 3rd, 4th and 5th freeze-thaw cycles showed significant (p<0.05) reduction in bilirubin level when compared with the baseline value. Serum bilirubin level has shown a strong negative correlation with time at higher temperatures than at lower temperatures [25°C (r=-0.943), at 4 °C (r=-0.493) and at -20°C (r=-0.355)]. Serum samples subjected to multiple freeze-thaw cycles showed decrease in bilirubin concentration with increasing number of freeze-thaw cycles (r= -986). The results indicated that serum can be stored for total bilirubin estimation without light exposure at 4°C and 25°C for at least 8 hours and at -20°C for at least 5 days. However, repeated freeze- thaw cycles significantly decreased the total bilirubin concentration after 2nd freeze-thaw cycle.