Effect of Multiple Freeze–Thaw Cycles on Coagulation Testing

International Council for Standardization in Haematology (ICSH) recommendations for processing of blood samples for coagulation testing

“…74 Differences between results obtained on samples stored in screw cap tubes with rubber seals and snap top versions were clinically irrelevant and either can be used. 74 Secondary aliquots for deep freezing should be capped throughout preparation, freezing and thawing. 74 When platelet-poor plasma (PPP) is frozen for future testing, the freezing and storage conditions affect the stability of frozen material.…”

“…74 Secondary aliquots for deep freezing should be capped throughout preparation, freezing and thawing. 74 When platelet-poor plasma (PPP) is frozen for future testing, the freezing and storage conditions affect the stability of frozen material. Rapid freezing reduces the opportunity for changes to the clotting factors and is more easily achieved when the secondary tube is transferred directly into storage units already present in the freezer for sufficient time to be acclimatized to the freezer temperature, rather than transferring samples in carriers or storage units that have been at ambient temperature.…”

“…If the volume of plasma is less than 1 mL, then 5 minutes thawing time is sufficient. 74 Thawing appropriately (at 37°C for 5 minutes) without post-thaw mixing is not sufficient since a lack of mixing was associated with a 60% loss of VWF activity and antigen from normal plasmas which was avoided by any of 3 different mixing methods, that is gentle mixing by 6-8 inversions;…”

“…81 A comprehensive study assessing seven cycles of freeze thaw (with −70°C storage) confirmed less than 5% change after two cycles for all parameters studied and <6% change after 3 cycles. 74 The authors studied PT, APTT, Fibrinogen, D-Dimer, clot-based FII/FV/FVII/ FVIII/FIX/FXI/FXII, chromogenic FXIII, AT activity, PC activity, PS activity plasminogen, DRVVT and anti-Xa (LMWH). Five freeze-thaw cycles did not cause significant changes in anticardiolipin or anti β2 glycoprotein 1 antibody testing.…”

Editorial Compilation VIII

“…Finally, Gosselin et al report on a small study on the effect of multiple freeze-thaw cycles on coagulation testing. 28 Although historically, it has been indicated in reagent manufacturer package inserts that plasma samples for coagulation should avoid multiple freeze thaw processes, since sample integrity may be compromised, solid evidence for any effects are largely lacking. Thus, this report should be of interest to laboratory personnel who otherwise may struggle on the number of "acceptable" freeze-thaw cycles for hemostasis testing, and this report provides specific insight into other preanalytical variables in coagulation testing.…”