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.
Additional references:
- Adcock DM, Mammen J, Nair SC, De Lima Montalvao SA. Quality laboratory issues in bleeding disorders. Haemophilia. 2016;22:84-89.
- Favaloro EJ, Pasalic L, Curnow J. Laboratory tests used to help diagnose von Willebrand disease: an update. Pathology. 2016;48:303-318.
- Sharma R, Flood VH. Advances in the diagnosis and treatment of Von Willebrand disease. Blood. 2017;130:2386-2391.
- Smith LJ. Laboratory diagnosis of von Willebrand disease. Clin Lab Sci. 2017;30:65-74.
I agree with Dr Gosselin that labs should in general follow reagent IFU’s.
In regards to “no FDA, so that [Australians] can do whatever they want Down Under”–well, I have written many ‘blogs’ on this*. Australian labs are highly regulated, albeit perhaps not to the level of US labs. Australian labs have to follow the ISO15189 standard, which was recently updated in 2022, as well as several NPAAC (National Pathology Accreditation Advisory Council) standards, and need to be accredited by NATA (National Association of Testing Authorities), which have recently adopted a risk-based model of accreditation. Laboratory practice is also in part controlled by our TGA (Therapeutic Goods Administration), the local equivalent to the US FDA. Part of the NATA accreditation assessment involves review of documentation, and part involves a peer review of processes by an active lab scientist or pathologist. Use of CLSI guidance, where relevant, is also in general encouraged. Not sure that counts for less ‘oversight’ per se than US labs. However, what appears to also be true is that oversight of labs in Australia is less ‘prescriptive’ than in the US. Thus, Australian labs have greater access to newer methodologies than US labs. As long as a reagent has TGA registration, it can be used in Australia as an in vitro diagnostic (IVD). Currently, the TGA essentially aligns to the European IVD regulation (CE-marked) process, although rumors related to tightening regulation continue to surface. I am not a fan of the US model. For a ‘free society,’ the US has a greatly regulated IVD process. Largely centered on a risk-based model, the process tends to assess the risk of introducing an IVD, but seems to ignore the risk of not introducing that IVD. For example, the US FDA has only recently cleared a modern alternative (VWF:GPIbM) to the classical ristocetin cofactor (VWF:RCo) assay for measuring von Willebrand factor activity; so, until recently, labs in the US only had FDA cleared VWF activity assays originally developed in the 1970’s (VWF:RCo) or an alternative monoclonal antibody based assay (VWF:Ab), which some argue is not a VWF activity assay. There is no VWF collagen binding (VWF:CB) assay FDA cleared, nor are VWF:GPIbR assays as yet cleared. Not sure how forcing labs to adhere to dated technology improves diagnosis of von Willebrand disease (VWD). As another example, despite the availability of current direct oral anticoagulants (DOAC) from the the 2010’s, there is still limited availability of DOAC measuring IVDs that are FDA cleared. Instead, Australian labs have greater access to modern methods of disease identification, and better ways to monitor efficacy of drug therapy than US labs. Not only are VWF:CB assays available, but several alternate VWF activity assays are also available, and all these represent current technology. Australian labs can also measure all the DOACs accurately. I know this because I am also very active in the local external quality assurance program (RCPAQAP) for hemostasis analytes. Early introduction of modern IVDs improves the accuracy of disease diagnosis and management. I cannot see how delaying introduction of modern IVDs for decades helps to achieve this.
* Favaloro EJ. Standardization, regulation, quality assurance and emerging technologies in hemostasis: issues, controversies, benefits, and limitations. Semin Thromb Hemost. 2007 Apr;33(3):290-7. doi: 10.1055/s-2007-971816. PMID: 17427064
Favaloro EJ. Harmonising quality to the lowest clinical diagnostic standard? The case against regulation of in vitro diagnostics (IVDs) for use in clinical diagnostic laboratories. Australian Journal of Medical Science, 2010; 31:56-64
Favaloro EJ. Regulation of in vitro diagnostics (IVDs) for use in Australian pathology laboratories: A gloomy outlook for future pathology testing in this country? Pathology. 2011 Jun;43(4):397-402. doi: 10.1097/PAT.0b013e3283468c70. PMID: 21566504.
Favaloro EJ, Plebani M, Lippi G. Regulation of in vitro diagnostics (IVDs) for use in clinical diagnostic laboratories: Towards the light or dark in clinical laboratory testing? Clin Chem Lab Med. 2011 Aug 30;49(12):1965-73. doi: 10.1515/CCLM.2011.690. PMID: 21875399
Favaloro EJ, Plebani M, Lippi G. Regulation in hemostasis and thrombosis: part I-in vitro diagnostics. Semin Thromb Hemost. 2013 Apr;39(3):235-49. doi: 10.1055/s-0033-1336833. PMID: 23483453
Favaloro EJ. Comparing the quality of testing for von Willebrand disease in different geographic localities. Haemophilia. 2022 Jan 31. doi: 10.1111/hae.14499. Epub ahead of print. PMID: 35099101
Favaloro EJ, Pasalic L. Laboratory Diagnosis of von Willebrand Disease (VWD): Geographical Perspectives. Semin Thromb Hemost. 2022 Sep;48(6):750-766. doi: 10.1055/s-0042-1754331. PMID: 36055264
George, there is an updated H21 that just came out with revised numbers for sample storage stability. That said, refer to the reagent IFU as they likely have stated stabilities. If one goes beyond the documented limits, then the assay is likely considered an LDT and must be locally validated. Dr. Favaloro has no FDA, so they can do whatever they want Down Under! About post-thawing, FDA IVD submissions for coag testing must be completed within 2 hours of thaw. [likely based on folklore passed down through the ages by the elders.]
[Note from Geo, I look forward to reading Dr. Favaloro’s response.]
Oh, in terms of how soon to run an assay after thawing; again, depends on the analyte. In general, I would say within 1 hour of thawing, but if there is a large batch of samples to assay, the testing time may potentially exceed 1 hr, so I always go with “as soon as possible”!
Hi George, yes, the CLSI guide seems to be the major resource for this question. I was involved in some updates (Lippi G, et al. Quality Standards for Sample Collection in Coagulation Testing. Semin Thromb Hemost. 2012;38:565-75. doi: 10.1055/s-0032-1315961; Adcock et al. Quality Standards for Sample Processing, Transportation, and Storage in Hemostasis Testing. Semin Thromb Hemost. 2012;38:576-85. doi: 10.1055/s-0032-1319768), but not really aware of any more updated evidence. In our lab, we initially store in a -20C freezer, and move items into a -80C freezer for longer-term storage. Most of our special tests have a <1 week turnaround, so they don't stay in the -20C freezer for long. I suspect that many analytes would survive storage at -20C for longer than 1 week, but some analytes do not, and so we tend to harmonize to the worse case scenarios. Certainly, factor assays could be compromised if stored for longer than 1-2 weeks at -20C. I would encourage someone to dig further and advise the readers of any newer evidence that we've missed or perhaps even to do an updated study. Of course, such a study may be biased to a particular -20C freezer, and as you point out, not all freezers are alike.
For Heparin specimens in my experience at 2 different Trauma level 1 academic medical center laboratories, heparin specimens were double spun to obtain platelet-poor plasma, and stored in a -70C freezer within 30 minutes of obtaining the specimens. If the initial option is to store only in a -20C freezer, then the specimens were only stored for a maximum of 7 days. Although not published, I have seen some discrepancies if heparin specimens that were stored in -20C were run after 7 days. After thawing at 37C, samples were tested ASAP without any delay. The total time from sample thawing and ending test run was not more than 20 minutes. If there were multiple specimens, then batches of 3-5 specimens were thawed so that any thawed specimens were not sitting on the bench for a prolonged time to be analyzed.