Effects of temperature and light on the stability of bilirubin in plasma samples

Although it is known that bilirubin is photo-sensitive, detailed effects of both temperature and artificial light exposure on bilirubin stability in plasma have not been well investigated. We determined the impact of temperature and artificial light on bilirubin stability in plasma.

Plasma total and direct bilirubin were analyzed using a diazo method. The aliquots of 38 samples were stored at 3°C and 22°C with light protection for 2, 4, 8, and 24h respectively before analysis. The aliquots of 20 samples with normal bilirubin and additional 20 with elevated bilirubin were exposed to artificial light for 2, 4, 8, 24, and 48 h at 22°C, and total and direct bilirubin were measured. The differences between the baselines and subsequent measurements were analyzed with analysis of variance.

The baseline total bilirubin was 9.6±8.1 mg/dl (mean±SD) and the concentrations were 9.6±8.2, 9.0±7.4, 9.0±7.5, and 8.8±7.5 mg/dl at 3°C and 9.5±8.1, 9.0±7.4, 9.6±8.1, and 9.5±8.0 mg/dl at 22°C after 2, 4, 8, and 24 h (p>0.05, n=38). The baseline direct bilirubin was 1.3±1.2 mg/dl and the concentrations after 2, 4, 8, and 24 h were 1.4±1.2, 1.4±1.2, 1.5±1.2, and 1.3±1.1 mg/dl at 3°C and 1.4±1.1, 1.3±1.1, 1.3±1.1, and 1.3±1.0 mg/dl at 22°C (p>0.05, n=19). In samples with elevated bilirubin exposed to light at 22°C, the baseline total and direct bilirubin concentrations were 10.2±1.7 mg/dl and 5.0±1.9 mg/dl, respectively. After 2, 4, 8, 24, and 48 h, total bilirubin concentrations were 10.1±1.8, 10.0±1.8, 10.0±1.8, 9.3±2.0 (p>0.05, n=20), and 8.4±2.3 (p<0.01, n=20) mg/dl and direct bilirubin concentrations were 4.9±1.8, 4.9±1.9, 4.8±1.8, 4.2±1.6 (p>0.05, n=20), and 3.5±1.5 (p<0.01, n=20) mg/dl. For samples with normal bilirubin levels under the same conditions, the average baseline total and direct bilirubin concentrations were 0.7±0.1 mg/dl and below the lower limit of quantification (LLOQ), respectively. After 2, 4, 8, 24, and 48 h, the average total bilirubin concentrations were 0.7±0.1, 0.6±0.1, 0.6±0.1 (p>0.05, n=20), 0.5±0.1, and 0.4±0.1 mg/dl (p<0.01, n=20) and direct bilirubin concentrations were still below LLOQ.

Bilirubin in plasma is stable in refrigerator or at room temperature without light exposure for at least 24 h. In normal laboratory environment, a delay of up to 8 h in the measurement of bilirubin left unprotected from light at room temperature does not significantly affect the results. Under these conditions, the changes in bilirubin concentration are not clinically significant until 24 h (direct bilirubin) and after 48 h (total bilirubin).