There is no point in collecting precious specimens only to find that they have deteriorated while in biobanking cryostorage. However, it is not just storage that can depreciate sample quality: shipping can also affect final quality. Posevitz-Fejfár et al. (2014) looked at the effect of transportation, focusing on biobanking peripheral blood mononuclear cells (PBMCs) from healthy human volunteers.1
For multicenter and long-term studies, it is essential that materials collected be consistent and represent normality. For this reason, optimized and standardized collection and subsequent biobank storage practices reduce variability. Collecting samples from remote locations away from a centralized biobanking repository and research center introduces risks from transportation that may not always be apparent when the samples undergo analysis.
Immune cell biobanking presents its own set of variabilities that could potentially skew study data. It is essential that the PMBCs collected are conserved in a natural state that renders them functionally unaltered for study. They need to be both viable for growth in culture and phenotypically capable of a valid response to immune stimulation.
With previous studies indicating altered immune cell function in response to different shipping and handling processes,2 Posevitz-Fejfár et al. collected blood samples from nine healthy volunteers at both a central repository (no shipping) and from a remote location (shipping required). An express courier firm shipped the remote samples to the central location within 24 hours, transporting the unprocessed samples under ambient temperatures overnight. The research team processed the samples within two hours of collection (no shipping) and within two hours of arrival (shipped). They processed the samples using standard methodologies for harvesting and cryopreserving PMBCs, using a Mr. Frosty device (Thermo Scientific) to bring the cells down to -80°C before storing them in the vapor phase of liquid nitrogen.
Prior to starting the main study, the researchers compared various shipping tube products and found no influence on the parameters under investigation. Comparing the two sets of samples, they did not find any difference in cell yield and viability with shipping. However, the shipped cells showed reduced viability and yield following recovery from cryopreservation, although the differences were not significant. The research team concluded that cryopreservation inflicts some damage on cells, which could explain this observation.
Using standard protocols to thaw the samples, Posevitz-Fejfár et al. assessed cell viability and function using proliferation and cytokine assays in addition to examining T cell receptor (TCR) and cytokine receptor signaling.The team found differences in immune phenotyping between the two sets, with an altered CD4+/CD8+ ratio, although this was not significant in the sample sizes examined. Levels of CD4+ cells increased, whereas CD8+ levels decreased. They found no significant phenotypic alteration in B cells or NK cells. Functionally, the team saw no significant alterations in the cells; cytokine secretion showed some sensitivity to shipping, whereas they found TCR and IL-6 mediated signaling functionally intact with only minor reductions. They saw a decrease in STAT3 phosphorylation with shipping.
Commenting on the sample size as a possible reason for the non-significant changes observed, Posevitz-Fejfár et al. advise caution in interpreting results when shipped PMBCs are included in multicenter studies. They advise cryopreservation at point of collection prior to shipping, or where possible, collection of all samples at a central biobanking repository. Where these steps are not possible, the researchers suggest incorporating blood samples from healthy donors as controls for the effects of transportation, as shipping does seem to alter PBMC quality.
For information on sample cooling rates, check out Fisher Bioservices White Paper: Controlled Rate Freezing of Cells During Ultra Cold Transit . It is comparative study that shows how staff at any clinical site can transport fresh PBMCs in -195°C in just 3 hours (including freezing).
1. Posevitz-Fejfár, A., et al. (2014) “Effects of blood transportation on human peripheral mononuclear cell yield, phenotype and function: Implications for immune cell biobanking,” PLoS ONE 9(12): e115920. doi:10.1371/journal.pone.0115920