Evaluation of Serum Volume Losses During Long-Term Storage

Aliquots of serum collected in a large case-control study of cervical cancer were stored at -70°C for up to 4 years during implementation of the study. When 500 μL serum aliquots were thawed in preparation for carotenoid and vitamin A assays, volumes were noticeably variable and fell below 500 μL in the majority of the samples. We were concerned about evaporation/sublimation during storage of the samples because loss of water would concentrate the analytes of interest. We evaluated the use of density and sodium ion concentration measurements to confirm its occurrence. We found that serum density was an unreliable indicator of extent of volume loss since the anticipated increases in density due to evaporation were of the same magnitude as inter-individual variation in serum density. In contrast, Na⁺ concentration is tightly regulated and would rise if water had been lost from the samples. In a representative sample of serum aliquots from the case-control study, 24 of 25 vials contained less than 500 μL of serum. The mean sodium ion concentration (138.1 ± 3.6 mmol/L) was within the normal range for human serum of 136-145 mmol/L, and no correlation was observed between serum volume and Na⁺ concentration. These results strongly suggest that the observed low volumes were not due to evaporative losses. Instead, the variably low volumes of serum aliquots were probably due to pipetting errors in the initial aliquotting resulting from the use of air-displacement pipettes.