The value of your work lies in the integrity of your specimens. Ensuring samples are maintained at the proper temperature throughout all processes is imperative to maximal long term viability and usability. This means not only during the processing and preparation of samples for analysis or storage—it also means during the transportation process. Moving samples between physical locations is a necessary part of biobanking, whether the locations are separated by a lab, a building or even a continent.
With ISBER 2016 right around the corner, let's take a trip down memory lane and review last year's workshop, presented by Alex Esmon PhD, global cold storage product manager at Thermo Fisher Scientific, on the importance of maintaining the cold chain during sample transport.
Last year's workshop focused on three different scenarios:
- Importance of maintaining sample conditions
- Concerns during inventorying
- Sample transport (unit transfer vs. content transfer)
Maintaining Sample Integrity
Ensuring sample integrity is paramount during any shipping or inventory activity. What's the point of shipping or inventorying samples that aren't worth saving? These processes can be accomplished in harmony but it's imperative to understand some basics about the freeze-thaw and ice phase transition process.
Refrigerated Storage: The +4°C Range
For refrigerated storage (+4°C temperature range) samples need to avoid freezing. Transitioning back and forth between frozen and thawed states is very bad for refrigerated samples. Ice formation and cellular dehydration are very stressful and will cause mechanical damage to biological samples. To avoid this, sample temperature should be maintained in the +10°C to +1°C range if possible. Here are some guidelines to follow:
- Guard against samples getting too warm
- Avoid dry ice for non-frozen samples
- A water/ice slurry is the best option for holding samples during the inventory process
- Minimize the duration and frequency of freezer door openings. This will help the samples that are not actively being handled to maintain their integrity and keep the samples in the right temperature range
Frozen Storage: The -20°C Range
Where refrigerated samples need to avoid freezing, frozen samples need to avoid thawing. The optimal storage temperature range is -20°C to -30°C. The key is to keep samples from thawing into the liquid state. The phase change from solid ice to liquid and back again is stressful for biological samples. Ice formation can perforate cell walls or change the structure of compounds. Below are some helpful guidelines:
- With samples in -20ºC freezers you have some options, but speed is the key
- Dry ice or bench-top coolers around -20ºC are better than just an ice bucket
- If using dry ice, be aware that enzymes in glycerol are not recommended to be frozen, so you must work quickly
- If an ice bucket is your only option, use a water and ice slurry for better temperature transfer than ice alone
Ultra Cold Storage: the -80°C Range
Thawing refers to not only the change from ice to liquid, but also the change from ice phase to ice phase. Keep in mind - looks can be deceiving. If a sample "looks frozen" that doesn't necessarily mean that changes aren't happening inside the sample. Samples stored at Ultra Cold temperature should be kept in the -60°C to -90°C if possible.
Samples in the ultra cold storage range toe the line between two forms of ice. As much as possible, these samples need to be handled using dry ice, which has a temperature of about -70°C. This will keep the samples from warming to a higher temperature range and will reduce undue stresses on the samples. If these samples thaw, it's important that a controlled freezing technique or equipment designed to control the rate of freezing is used to bring them back to the cryogenic range.
Samples stored at cryogenic temperatures should be kept at -130°C and below. Samples stored at this temperature range are in a "glassy state." They have crossed the glass transition phase and have taken on the properties of glass. Below this threshold, all known mechanical and metabolic activity within the cells stops.
It is imperative to keep samples that are stored below the glass transition phase under this threshold at all times. Samples that toggle back and forth across this threshold can experience instantaneous ice nuclei formation which can seriously damage or destroy samples.
In summary, frozen samples need to avoid thawing, refrigerated samples need to avoid freezing, and if thawing does occur, be prepared with suitable re-freeze plans to account for the needs of the samples.