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Freezing Coagulation Specimens

From Maureen Caraker:
If coagulation testing is not performed immediately and needs to be prepared for send outor stored for special coagulation batch testing, is it necessary to flash freeze plasma? I cannot find any regulation addressing this.
Hi, Maureen, thank you for your question. H-21 A5 is the current CLSI standard for coagulation specimen, and it requires that specimens which cannot be tested within the specified limits should be centrifuged to be platelet poor (≤10,000 platelets/µL) and frozen. The standard does not use the phrase “flash-frozen” but does recommend placing the specimen in a −70° C freezer if one is available. If only a −20° C freezer is available, it is acceptable. The specimen absolutely must be platelet poor. Geo.

Comments (3)
Oct 7, 2009 2:55am

George and David: This is an interesting question and thank
George and David: This is an interesting question and thanks to David for citing his study. Perhaps there is a subtle distinction between “frozen at” vs. “storage at” for plasma samples? I noted in David’s study that the -70C samples were snap-frozen in liquid N2 (-196C) while the samples stored at -20C samples were frozen at -20. In our experience, the critical point influencing plasma coag factors occurs around the phase-change of the plasma (i.e. from liquid to solid and vice-versa) Slow freezes at -20C can compromise protein structure due to influence of ice crystal formation. This is substantially mitigated by snap-freezing at very low temperatures. On the other hand, slow thawing can lead to cryoprecipitation and low Fibrinogen, vWF and FVIII:C levels. It would be interesting to repeat David’s study and control for these two variables (freezing temp and storage temp). My two cents! — Steve

Oct 2, 2009 8:24am

Thank you, Dave. I stand corrected regarding the -20 degree
Thank you, Dave. I stand corrected regarding the -20 degree C freezer. I have the 2007 edition of CLSI H21-A5 and they do allow 2 weeks’ storage at -20, warning that if the specimen is stored longer it must go to a -70 freezer. The H21 authors need to know about your study; I didn’t find it referenced there! Also, what I don’t see in H21 is any reference to flash- or snap-freezing. H21 just says to place the specimen in the freezer. Also, we haven’t completely answered Maureen’s question as there is no actual definition for flash- or snap-freezing. In your study you used liquid nitrogen, which is the coldest thing around and could surely fit the definition if there was one, but most of us only have access to -20 or -70 freezers. Would placing the specimen in a -20 freezer be considered flash-freezing? Don’t know, and I suspect it partly depends upon the specimen volume. I’m hoping Dr. Adcock or one of the other H21 authors sees this and extends our conversation. Geo.

Oct 1, 2009 7:24am

I have to disagree with the comment of -20 degree C being ac
I have to disagree with the comment of -20 degree C being acceptable for storing platelet-poor-plasma for coagulation testing. In a study conducted long ago and published in the TEXAN, the Texas Journal of Medical Technology 1987;4:10-11, I found that -20 was not sufficient for specimen storage. I drew specimens from normal male volunteers and obtained citrated platelet poor plasma. I then aliquoted samples and snap froze using liquid nitrogen one set, and stored them at -70C, a second set was frozen at -20C, and a third set of samples was analyzed within 90 minutes of collection. The frozen specimens were tested after 5 days of storage. I analyzed each sample in each group for PT, APTT, FVIII:C FVIII:Ag and FVII. The FVIII:C activity assay was the only parameter that was significantly different (p<;0.05). A loss of 50.4% mean activity was evident in the -20C samples when compared to the baseline activity level of the unrefrigerated specimens. It was even more when compared to the -70C group(67.8% activity). I determined that storage of samples for coagulation testing at -20C was not sufficient for specimen storage when analysis for a thermolabile factor such as FVIII:C is required. Therefore, why possibly compromise a specimen for coagulation testing by storing at a temperature that can’t preserve all coagulation factors. Storing at -70C is the best option.

David L. McGlasson, MS, MLS
59th Clinical Research Division
Lacklnad AFB, TX

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