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Flash-freezing in IPA/Dry Ice

Here is a challenging question from Anne-Marie Quinn, Janssen-Ortho Canada:

Hi, I am starting up a PK study at a new facility that has not done flash freezing of plasma samples before. I want them to flash freeze in an isopropyl alcohol-dry ice bath, but I am unable to find any standard method that specifies how long the samples need to be in the bath before they are considered to be flash frozen. I have found a method for ascorbic acid in plasma that requires 2 hours in a -70°C freezer, but I am thinking that the full 2 hrs are not necessary. Do you happen to know a reference that describes the time required at -70°C to bring 0°C plasma to -70°C?

Thanks in advance.

Hello, Anne-Marie, and thank you for your question. Like you, I have found no reference that provides specific instructions for flash-freezing specimens, including a search through Clinical and Laboratory Standards Institute (CLSI) specimen-management guidelines and standards. So, in the absence of facts, I will do a little speculating, then will turn it over to our participants for their experiences.

First, I am curious about the need for flash-freezing. You mention “PK,” which I interpret as prekallikrein. If this is your analyte, there is no need for flash-freezing, as prekallikrein is stable at room temperature. From our subsequent e-mail discussion, not reproduced here, I conclude your analyte may be something other than prekallikrein.

Second, it appears from your question (and our subsequent discussion) that you chill your specimens prior to separation and freezing. While chilling may be apropos to your situation, we avoid chilling coagulation specimens as it causes precipitation of large von Willebrand factor multimers, release of platelet granule materials such as platelet factor 4, and activation of factor VII, all documented phenomena. Clinical standards such as CLSI’s H21-A5, require that coagulation specimens be managed at ambient temperatures.

The above issues aside, if your protocol does require flash-freezing, I know of no data specifying time in the freezing bath, volume of the aliquot being subjected to freezing, or temperature achieved within the aliquot once frozen. I think it is safe to assume, however that the goal is near-instantaneous freezing, which implies a small aliquot (perhaps less than 1 mL) and a storage vial designed to withstand cold temperatures.

If I may express another opinion, it may be that the institution you are working with has access to liquid nitrogen freezers. These would be preferable to IPA/dry ice as they would be safer, cleaner, more standardized, and would provide both freezing and storage in a single-step process. Whereas the temperature of an IPA/dry ice bath would vary by the volume of IPA and dry ice and preparation time, liquid nitrogen stays at a reproducible -196°C.

Long-term storage at -196°C is the most reliable, but if it is not available, I’d recommend a -70°C freezer. As you mention in our correspondence, a household freezer, which maintains approximately -20°C, is somewhat less reliable as the temperature rises whenever someone opens the door to obtain a specimen. Most household freezers provide a defrost cycle, which would be undesirable for specimen storage.

Finally, it may also be that placing your aliquots in a -70°C freezer fulfills the requirements for flash-freezing. Again, if the specimen is small enough, freezing in a -70°C freezer should be instantaneous.

That’s all I have, let’s see what responses we receive from other Fritsma Factor participants. Geo.

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