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Letters to the Editor |
Sezione di Chimica, e Microscopia Clinica, Dipartimento di Scienze, Morfologico-Biomediche, Università degli Studi di Verona, Verona, Italy
aAddress correspondence to this author at: Istituto di Chimica e Microscopia Clinica, Dipartimento di Scienze Morfologico-Biomediche, Università degli Studi di Verona, Ospedale Policlinico G.B. Rossi, Piazzale Scuro, 10, 37134 Verona, Italy. Fax 39-045-820-1889; e-mail ulippi{at}tin.it.
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 hemostatic system. By using a fluorogenic substrate, the TGA produces thrombin-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 
1.5 min indicates the need for factor V Leiden genotyping, whereas a peak thrombin concentration >433 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 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).
Because the freezing-thawing effects on ETP, lag time, and cmax cannot be anticipated and depend on heterogeneous interindividual functional characteristics of platelets(7), the use of thawed PRP is likely to introduce an unpredictable bias, influencing result comparability and transferability within the same study protocol, especially during assessment of the APC-induced thrombin potential inhibition(2). Additionally, although the use of frozen PRP may be justified to screen for the presence of lupus anticoagulants(3), there are no clear reasons to use PRP to screen for inherited coagulation disorders that do not directly involve platelet pathophysiology, such as factor V Leiden and the factor II G20210A sequence variant(8).
In conclusion, we acknowledge that, on the whole, the TGA might be potentially useful for the laboratory assessment of a large spectrum of clotting abnormalities. Nevertheless, as with other areas of coagulation testing, we suggest that rigorous preanalytic and experimental conditions for the TGA ought to be fulfilled and standardized(2)(3)(4)(6) to provide reliable information on clinically meaningful hypercoagulable states.
References
The following articles in journals at HighWire Press have cited this article:
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  N. Hezard, M.-G. Remy, B. Florent, and P. Nguyen Reliability of Thrombin Generation Assay on Frozen-Thawed Platelet-Rich Plasma: A Reply. Clin. Chem., November 1, 2006; 52(11): 2127 - 2128. [Full Text] [PDF]
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