Reliability of the thrombin-generation assay in frozen-thawed platelet-rich plasma.

To the Editor: The recent introduction of a general coagulation function test, namely the thrombin-generation assay (TGA), has enabled efficient assessment of the global functioning of the hemo-static system. By using a fluorogenic substrate, the TGA produces throm-bin-generation curves in a fully automated manner that may be useful and sensitive enough to screen for either hypercoagulable states or hemorrhagic diatheses. In the recent report, Hézard et al. (1), concluded that the TGA can be reliably used to screen patients needing further specific thrombophilia testing. Specifically , a thrombin generation lag time Յ1.5 min indicates the need for factor V Leiden genotyping, whereas a peak thrombin concentration Ͼ433 nmol/L indicates the need for factor II G20210A genotyping. As reported in that study, the experiments were performed on thawed, previously frozen, platelet-rich plasma (PRP), and little indication is provided on either the collection procedure or the storage conditions of these samples, both of which are essential requisites to enable reliable TGA results (2, 3). Previous exhaustive evaluations of TGA demonstrated that although the integral amount of thrombin generated in time, expressed by the endog-enous 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 procoagu-lant phospholipid exposure (2, 3). Accordingly, in frozen-thawed PRP, the lag time decreases substantially, up to one third, compared with non-frozen specimens (2). The freezing also affects the maximum concentration of thrombin (c max), 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. Plate-lets 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 …