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Freezing and the Thrombin Clotting Time

This is a message from Mary Dugal, Precision BioLogic Inc Account Manager:

During a recent meeting, a customer of mine described the following anomaly and I’m posting this question to you on her behalf.

“When performing comparisons with the intention of switching to a different concentration of thrombin clotting time (TCT, TT) reagent, we noticed the results were consistently lower post-thaw on prolonged samples containing unfractionated heparin (UFH):  80 s versus 50 s; 89 s versus 34 s; and 42 s versus 24 s. Our normal TTs compared well.

We used double centrifugation of samples, froze at -70°C and thawed in a 37°C water bath. When correlating with several sites using the same instrument, reagent, and thrombin concentration, the same pattern was seen. Heparin assays were performed on samples and were stable pre- and post-thaw.”

Mary continues that her friend found an article published in 1960 where it states that plasma is sensitive to heparin, when frozen for more than 24 hours, which results in lower thrombin time values post thaw. Have you experienced this phenomenon in your practise? Many thanks in advance!

Hi, Mary, and thank you for your question. I have no direct knowledge of the effect of UFH on the TT, although I know the TT may be used to monitor UFH therapy and is preferred to the partial thromboplastin time (PTT) by some, particularly in Northern Europe. I’m guessing that despite all precautions, including double centrifugation, UFH is partially neutralized by platelet factor 4 during centrifugation, freezing, or thawing. Alternatively, UFH may deteriorate while frozen or in the thawing process. I’d like to hear from those who use the TT to monitor heparin, you may have more experience with this phenomenon. Geo.

Comments (1)
Jul 25, 2011 10:29am

I posed this question to Dr. Alvin Schmaier, who responded:
I posed this question to Dr. Alvin Schmaier, who responded: We used to monitor heparin with the TCT at Michigan. However, we always ran it on a freshly obtained sample. I do not recall data on fresh-frozen and then thawed samples. I could propose various mechanism(s) for the shortening of the thawed sample, but without experimentation, I would not know the precise one.

Alvin H. Schmaier, MD
Robert W. Kellermeyer Professor of Hematology/Oncology
Case Western Reserve University
University Hospitals Case Medical Center
Cleveland, OH 44106-7284

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