Biobank and biorepository staff members, in order to ensure and promote sample integrity, may need to caution investigators about the potential effects of dry ice on the samples they receive. Depending on the type of assay to be performed on the sample, recipients may need to allow the sample to “normalize” after being removed from the shipper rather than being allowed to thaw “as is.” Samples shipped on dry ice are exposed to CO2, which causes a drop in pH, which then results in changes to the properties of the proteins in the sample.
CO2 exposure, and the resulting drop in pH, can alter protein structure, solubility, and multiple other characteristics that will skew assay results. If you have been getting erratic data from samples that were shipped to you in a frozen state, the culprit may be the dry ice. The concentration of CO2 inside a shipper can readily exceed 2,500 µg/ml.
Some containers and lids are more permeable than others to CO2—these include silicone rubber and polypropylene—but no container will prevent entry of CO2. The issue is generally not the container, but is typically the stress placed on the seal at ultra-low temperatures. Seals lose some degree of their integrity at -70°C, and because the loss in integrity varies unpredictably, the change in pH resulting from differences in CO2 exposure also varies unpredictably.
The good news is that this influence is transient. According to data published by Murphy et al in the April 2013 issue of Nature Methods [Murphy, B.; Swarts, S.; Mueller, B.; van der Geer, P.; Manning, M. & Fitchmun, M., 10, pp. 278–279 (2013)], there are two ways to prevent this phenomenon from affecting your assay from protein instability following transport or storage on dry ice.
The first is to allow the samples to normalize in an ultra-low freezer for 96 hours prior to performing your assay, to allow the CO2 to dissipate. If the sample is not needed for several days, this is the easiest solution. However, if that particular analysis won’t wait a few days, the second solution is to remember to remove the cap and vent the CO2 before thawing the sample.
Any CO2 that leaks into the sample container will remain confined to the head space as long as the sample remains below -40°C. Rather, the drop in pH occurs as the sample warms, and the higher temperature allows the CO2 to interact with the proteins. The simple step of removing the cap will prevent this interaction.
Murphy et al. noted that although leaks in the seal between the container and the lid are most likely the source of the CO2 exposure, various attempts to protect the sample were not effective. His team tried wrapping containers with Parafilm, placing them in laminated aluminized Mylar zip seal bags, and also heat-sealing the laminated aluminized Mylar bags. The heal-sealed bags prevented acidification but the other two methods did not yield reliable results.
Sample integrity is a significant concern for biobanks and opportunities for affordable alternatives to shipping materials on. If you are interested in related information about cell therapy logistics, please check out my eBook, Commercially Successful Cell Therapies or recent video blog, Delivering Your Cell Therapy: Getting Reliable Performance from Dry Shippers.