Reliability of the Thrombin-Generation Assay in Frozen-Thawed Platelet-Rich Plasma

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Abstract
Previous exhaustive evaluations of TGA demonstrated that although the integral amount of thrombin generated in time, expressed by the endogenous thrombin potential (ETP), appears substantially unmodified in frozen-thawed PRP, thrombin generation is accelerated and the maximum amount of generated thrombin is increased, apparently as a result of cold-induced platelet activation, membrane damage, and procoagulant phospholipid exposure(2)(3). Accordingly, in frozen-thawed PRP, the lag time decreases substantially, up to one third, compared with nonfrozen specimens(2). The freezing also affects the maximum concentration of thrombin (cmax), which is substantially higher in frozen-thawed than in fresh PRP. Thus, it seems likely that assessing thrombin generation in frozen-thawed PRP would introduce a substantial bias in several measurements, especially lag time and peak concentration. Consequently, the ETP would appear to be the single variable that can be assessed in PRP, regardless of the storage conditions(2). However, this is further disputed by Chantarangkul et al.(4), who demonstrated that when the phospholipids are omitted, such as in the experimental conditions of Hézard et al.(1), there is a linear relationship between the ETP value and the number of residual platelets in thawed specimens. Platelets are not an ideal surrogate for exogenous phospholipids, as the fatty acid composition of membrane phospholipids in platelets might be heterogeneous, depending basically on dietary lipid modifications(5). Additionally, the interindividual variability of several TGA indicators measured in PRP is considerably higher, especially in the presence of very low concentrations (≤3 pmol/L) of tissue factor(2). Potential artifacts in thawed specimens, such as platelet debris or the presence of procoagulant material, are detrimental to assay reliability(2). Although ideally TGA should be evaluated on whole blood or PRP, we recommended that frozen plasma is suitable, provided that it is filtered before testing to eliminate the unwanted effect of residual platelets(6). Earlier data showed an increased sensitivity to activated protein C (APC) in frozen-thawed PRP compared with fresh PRP(3). To minimize the influence of using frozen-thawed PRP preparations, Regnault et al.(3) suggested that 6.7 nmol/L exogenous APC be added, instead of 25 nmol/L, the latter being the experimental conditions of Hézard et al.(1).