Yesterday, June 14, 2023, I [Geo] received two questions about coagulation plasma specimen management. Heather DeVries, Indiana University, asked if there were data supporting the CLSI two-week maximum for storing plasma frozen at –20°C, and Jessica Warner, Kettering Health, asked how soon after thawing you must run an assay. The CLSI guideline H21-A5, Clinical and Laboratory Standards Institute (CLSI). Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays (5th edn., H21-A5). Wayne, PA: CLSI; 2008, states that specimens may be stored frozen at –20°C for up to two weeks or 12 months at –70°C or colder. Frozen plasma aliquots must be thawed rapidly at 37°C, gently mixed, and tested immediately.
There is more detail. The CLSI21-A5 authors reference Woodhams B, Girardot O, Blanco MJ, Colesse G, Gourmelin Y. Stability of coagulation proteins in frozen plasma. Blood Coagul Fibrinolysis. 2001;12:229–36. The investigators collected plasma from six subjects using plasmapheresis, creating a pooled 1:9 dilution in 0.129 M sodium citrate. They tested several coagulation parameters over 24 months and found that they were stable for at least 3 months at –24°C and 18 months at –74°C. The first question came from a Medical Laboratory Scientist collecting specimens for chromogenic anti-Xa heparin assay validation. The Woodhams study used plasma from only healthy subjects, thus it could not be generalized to patient plasma containing therapeutic unfractionated heparin, leaving us with the original guideline.
There are some additional recommendations. We can only store plasma at –20°C in manually defrosted freezers, as most household freezers self-defrost by cycling between 0°C and –20°C, activating coagulation factor VII and causing VWF precipitation. Before freezing, specimens must be centrifuged [not filtered] to be platelet-poor, in which the plasma platelet count is less than 10,000/uL, as platelets secrete platelet factor 4, rapidly neutralizing unfractionated heparin.
For heparin specimens, and for practical purposes, all specimens, centrifugation, and separation must be completed and the aliquot stored in the freezer within one hour.
The guideline for thawing specimens is explicit and reflects the 37°C deterioration of factors V and VIII and control protein S, thus, the operator should be in a position to begin the assay as soon as the specimen thaws and is gently mixed.
We invite comments and your own potential modifications of these guidelines.
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