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.
