In contrast, other investigators have shown a loss of T-cell response to antigenic stimulation [31], while cryopreservation has been shown to induce apoptosis [16]. In addition, sample storage at −30 °C, or temperature rises mimicking sample transport conditions, have been shown to lead to a reduction in T-cell functionality [40] and cell damage [13]. Despite such investigations, there is a lack of data about the influence of temperature rises during sample storage,

sorting and removal, if specimens are cryopreserved in conventional liquid nitrogen tanks without a protective hood system to guard against temperature fluctuations. Our studies provide additional information that the quality of sample storage

and handling is critical for maintaining PBMC viability, 17-AAG PBMC recovery and T-cell functionality. Exposure of cryopreserved PBMC to suboptimal sample storage with repeated temperature fluctuations can lead to a reduction in sample quality. We have demonstrated that temperature shifts during storage reduce cell recovery and viability as measured by trypan blue dye exclusion and could resulted in significant cell death, especially after overnight culture. Other groups have also reported a reduction in cell viability after culture compared to immediately post thaw, suggesting that a population of cells still undergoes DAPT mouse apoptosis or necrosis following thawing [21] and [31]. Smith et al. (2007) showed an increase in the percentage of apoptotic cells after cyclical temperature rises and programmed cell death can be induced by physiological signals or by a number of physical events like

heat shock, free radicals, UV light and gamma radiation [43]. Cells can also receive signals that make them predisposed to apoptosis but they do not actually undergo cell death until the final signal is received [7], [8] and [17]. Suboptimal cryopreservation may prime the cells for the apoptotic pathway, without initiating the process. Overnight culture of the cryopreserved cells in the presence of mitogens, that are known 3-mercaptopyruvate sulfurtransferase to exist in fetal calf serum, could trigger the primed cells into the death cascade [13], [51] and [52]. We have also demonstrated that cyclical temperature rises during the storage process decrease T-cell functionality after stimulation with CEF and CMV peptide pools. Mimicking sample storage, sample sorting and sample removal processes that use a protective hood system increased the T-cell response by about 23% in comparison to the same procedures without protective hood system. The degree of reduction in T-cell functionality ranged from 0% to 74% and was donor-dependent and not predictable. For that reason it was not possible to apply a correction factor to the results received from the immune assays.