Thrombin generation and procoagulant phospholipids in patients with essential thrombocythemia and reactive thrombocytosis

Transcription

1 Research Article Thrombin generation and procoagulant phospholipids in patients with essential thrombocythemia and reactive thrombocytosis I. Mignon, 1 F. Grand, 1 F. Boyer, 2 M. Hunault-Berger, 2 J.F. Hamel, 3 and L. Macchi 1,4 * Thrombocytosis is a commonly encountered clinical scenario and can be either a secondary process (reactive thrombocytosis), or due to clonal disorder (i.e., essential thrombocythemia). This distinction is important as it carries implications for evaluation, prognosis and treatment. In this study we compared procoagulant potential in essential thrombocythemia and reactive thrombocytosis by measuring the thrombin generation and the level of circulating procoagulant phospholipids with functional tests. Twenty nine patients with essential thrombocythemia and 24 with reactive thrombocytosis were studied. Thrombin generation was determined by calibrated automated thrombography. Procoagulant phospholipids were detected by a chronometric standardised method (STA-Procoag-PPL). Patients with reactive thrombocytosis had a longer lag time, higher endogenous thrombin potential, peak of thrombin generation and velocity index than patients with essential thrombocythemia. The level of circulating procoagulant phospholipids was increased in patients with essential thrombocythemia as observed with the procoagulant phospholipids assay. Each parameter was analysed using ROC curves. Highest areas under the curve (AUC) were found for lag time and procoagulant phospholipids ratio (0.817 and 0.853, respectively), associated with high negative predictive value for ET (92.3% and 80%, respectively). In conclusion, patients with essential thrombocythemia and reactive thrombocytosis displayed significant differences in terms of thrombin generation and levels of procoagulant phospholipids. Among these parameters, lag time and procoagulant phospholipids ratio could help to differentiate between reactive thrombocytosis and essential thrombocythemia patients. Am. J. Hematol. 88: , VC 2013 Wiley Periodicals, Inc. Introduction Although the exact definition of thrombocytosis varies in the literature, it is generally defined as a platelet count exceeding /L in adults [1,2] Thrombocytosis can be either a secondary process, called reactive thrombocytosis (RT), or primary. Essential thrombocythemia (ET) is a chronic myeloproliferative neoplasm (MPN), found to be associated with the somatic V617F mutation in the Janus 2 tyrosine kinase (JAK2) gene in around 50% of cases [3 5]. The clinical course of ET is characterized by an increased frequency of thrombotic complications at venous, arterial, or microcirculatory sites, representing the leading cause of morbidity and mortality [6]. History of thrombosis and age over 60 are currently well-defined risk factors of thrombosis [7,8]. The origin of the hypercoagulable state in these patients is unclear and seems to involve numerous mechanisms. Platelet dysfunction [9] and the presence of activated circulating platelets, particularly in patients carrying the JAK2 V617F mutation [10 12] have been advocated. Leucocyte activation and the formation of platelet-leucocyte aggregates may also contribute to the thrombotic state in ET [12 14]. Some studies suggest a role for microparticles in thrombosis in ET. Microparticles are plasma membrane vesicles released by most cell types into circulation upon their activation or apoptosis, and are known to be elevated in thromboembolic diseases. In several studies, patients with ET had a higher number of circulating microparticles with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation [15,16]. RT can be found in various pathologic situations (i.e., infection, post operative period) and the differential diagnosis with ET is sometimes difficult. Thrombotic complications are less frequent in RT than in ET, but not rare [17]. Few studies were conducted to evaluate the procoagulant potential in the plasma of RT patients. One study indicates that the ratio platelet microparticles/platelet count was increased in ET compared with RT [18]. The main aim of our prospective observational study was to compare procoagulant potential in ET and RT by measuring the thrombin generation and the levels of VC 2013 Wiley Periodicals, Inc. American Journal of Hematology 1007 circulating procoagulant phospholipids with functional tests. Our secondary goal was to compare these biological parameters in ET patients according to (a) the JAK2 V617F mutation status and (b) to the presence of a medical history of arterial or venous thrombosis. Materials and Methods Study population From June 2009 to October 2011, 29 patients with ET (10 males and 19 females; age range: years; mean age: 68 years) and 24 patients with RT (13 males and 11 females; age range: years; mean age: 57 years) from the University Hospital of Angers were enrolled. ET was diagnosed according to the 2008 World Health Organization diagnostic criteria. RT was defined according to clinical and biological criteria obtained from hospital records. Patient hospital notes were also reviewed in order to obtain results of complete blood count and JAK2 V617F mutation positivity, to ascertain whether individuals had a medical history of arterial or venous thrombosis, and if they were taking any antiplatelet drug. Patients with RT were not taking any antiplatelet or antithrombotic drugs. All investigations were approved by the local ethic committee (Ethic Committee, University Hospital of Angers, France). Twenty healthy subjects (7 males and 13 females; age range: years; mean age: 36 years) acted as control group for thrombin generation test. None of them had taken treatment for at least two weeks, had symptoms of inflammation disease, or had history of thrombohemorrhagic events. 1 CHU Angers, Laboratoire d hematologie, Angers, France; 2 CHU Angers, Service d Hematologie Clinique, Angers, France; 3 CHU Angers, Centre de recherche clinique, Angers, France; 4 Universite d Angers Laboratoire Cardioprotection, remodelage et thrombose, Angers, France Conflict of interest: Nothing to report *Correspondence to: Dr Laurent Macchi, Laboratoire d Hematologie, CHU Angers, Angers, France. lamacchi@chu-angers.fr Received for publication 20 February 2013; Revised 8 July 2013; Accepted 15 July 2013 Am. J. Hematol. 88: , Published online 22 July 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: /ajh

2 Blood collection and plasma preparation Venous blood was taken by clean venipuncture with minimal stasis. Blood was collected into sterile siliconized tubes containing trisodium citrate (0.129 M, 1:9 vol:vol, Vacutainer, Becton Dickinson, Le Pont de Claix, France). Platelet-poor plasma (PPP) was isolated from citrated blood by double centrifugation at 3000g for 15 min at room temperature, and stored in 600 ml aliquots at 280 C until testing. Thrombin generation test (TGT) Thrombin generation was determined in PPP by calibrated automated thrombography (the Calibrated Automated Thrombogram). Eighty microliters plasma were pipetted into the well of a microtiter plate together with 20 ml of a mixture containing human recombinant tissue factor (5 pm) and synthetic phospholipid vesicles (4 mm) (PPPreagent 5 pm, Thrombinoscope BV, Maastricht, The Netherlands). The reaction was triggered with 40 ml of a mixture of fluorogenic substrate and calcium chloride (FluCa-Kit, Thrombinoscope BV, Maastricht, The Netherlands). Fluorescence was read in a Fluoroskan Ascent reader (Thermo Labsystems, Helsinki, Finland). The fluorescence signal was measured at 15 sec intervals over a period of 60 min. Thrombin generation curves were calculated using Thrombinoscope Software version (Thrombinoscope, Maastricht, The Netherlands). Thrombin generation curves were described in terms of lag-time, peak, area under the curve (known as the endogenous thrombin potential or ETP), and velocity index. Thrombin generation and sample specific calibrators were tested in triplicate for each sample. Procoagulant phospholipids assay Procoagulant phospholipids were detected in PPP by a chronometric standardised method (STA-Procoag-PPL, Stago, Asnières, France) with the analyser STA-R (Stago, Asnières, France). This test consisted of the measurement of clotting time in the presence of calcium, of a system in which the addition of phospholipids depleted plasma made the test dependant on the procoagulant phospholipids of the tested sample. This time was compared with a reference time. The final result could be expressed in clotting time, or in ratio (clotting time of the plasma being tested/reference clotting time). A shortening clotting time of sample compared with usual values of control plasma (ratio <1) meant an increase in procoagulant phospholipids. The reference clotting time was determined by the clotting time of a pool of at least 20 individual normal plasma samples, used undiluted and in the same conditions as the plasmas to be tested. Statistical analysis Quantitative data were reported as mean 6 standard deviation, and qualitative data as percentage. Data were compared using nonparametric tests: Kruskal-Wallis rank sum test for quantitative data and Fisher s exact test for qualitative data. For all analysis, a two-tailed test was used and P < 0.05 was considered significant. All statistical analyses were performed using Stata software (StataCorp, College Station, TX 77845). ROC curves were used for evaluating the diagnostic values of lag time, ETP, peak height, velocity index and clotting time ratio for ET. Parameters with lower bound of the AUC 95% confidence interval CI greater than 0.7 were considered as relevant for the ET diagnosis. For each parameter satisfying this criterion, we defined a cut-off maximizing the percentage of well-classified thrombocytosis. Then, we estimated positive and negative predictive values (PPV and NPV) associated with this cut-off. Results Characteristics of the study population The study population consisted of 53 patients with thrombocytosis, including 29 patients with ET and 24 patients with RT. Demographic and biological data are presented in Table I. Patients with ET were older than patients with RT (P < 0.05). Sex ratio between the two groups of patients was not significantly different (Table I).White blood cell and neutrophil counts were significantly lower in ET patients than in RT, whereas platelet count and hemoglobin were significantly higher (Table I). Considering ET patients, 21 (72%) were positive for the JAK2 V617F mutation and six (21%) had a history of thrombosis: four with arterial thrombosis (14%) and two with deep venous thrombosis (7%). None of the RT patients had thromboembolic medical history. At the time of enrolment, four ET patients (14%) were TABLE I. Demographic Data RT ET P N Male/female 13/11 10/ Age, years (6 SD) <0.05 Platelets, /L (6 SD) <0.001 White blood cells, /L (6 SD) <0.01 Neutrophils, /L (6 SD) <0.05 Haemoglobin (g/dl) (6 SD) <0.001 JAK2 V617F mutation positivity N/A 21 (72%) History of thrombosis 0 6 (21%) Arterial * 0 4 (14%) Venous # 0 2 (7%) Antiplatelet therapy 0 4 (14%) N/A, not applicable. Data presented as number (%) for sex, JAK2 V617F mutation, history of thrombosis and antiplatelet therapy. Data presented as mean (6SD) for the other parameters. RT, reactive thrombocytosis; ET, essential thrombocythemia. * Arterial thrombosis: acute coronary syndrome, transient ischemic attack, peripheral arterial occlusive disease. # Venous thrombosis: deep venous thrombosis (Kruskal-Wallis rank sum test and Fisher s test, P < 0.05). receiving antiplatelet therapy (two with aspirin alone, and two with aspirin plus clopidogrel). Regarding the etiologies of RT, about one third (33%) of the thrombocytosis was related to inflammation or infection, 8% of patients had both inflammation and potential drugs which could explain thrombocytosis, and 17% of patients were in a post-operative or post-traumatic context. Another cause was found in 30% of cases (alcohol withdrawal: 4%, cancer: 13%, iron deficiency: 13%). In 12% of patients, no etiology was found. Thrombin generation in essential thrombocythemia and reactive thrombocytosis Data are presented in Figure 1. Patients with RT had a longer lag time ( min) than ET patients ( min; P < 0.001) and controls ( min; P < 0.001, Fig. 1A). ETP was higher in RT patients (1, nmxmin) than ET patients ( nmxmin; P < 0.05, Fig. 1B) whereas it was lower in ET patients than in controls (1, nmxmin; P < 0.05). The difference in ETP between RT and controls was not significant. A similar significant difference was observed for the peak height of thrombin generation curves between RT patients ( nm) and ET patients ( nm; P < 0.001), and between controls ( nm; P < 0.05) and ET patients (Fig. 1C). The velocity index of thrombin generation was higher in RT patients ( nm/min) than in ET patients ( nm/min; P < 0.001) (Fi. 1D). The same significant differences of TGT parameters were observed between ET patients who were not taking antiplatelet drugs (n 5 25) and RT patients (data not shown). Procoagulant phospholipids in essential thrombocythemia and in reactive thrombocytosis The procoagulant assay was performed in 28 patients with ET and 16 patients with RT (Fig. 2). The value of the reference clotting time established in our laboratory from 20 normal plasma samples was 59.2 sec. The mean clotting time ratio was significantly lower in ET patients than in RT patients ( vs ; P < 0.001) reflecting a higher level of circulating procoagulant phospholipids in ET patients group. No correlation was found between leukocyte count and amount of procoagulant phospholipids. Thrombin generation and procoagulant phospholipids in ET patients according to JAK2 V617F mutation status and history of thrombosis No significant differences were found between JAK2 V617F - positive and -negative patients in term of lag time, peak, ETP, and velocity index (Table II). The TGT parameters were not significantly different between patients with and without 1008 American Journal of Hematology

3 Figure 1. Comparison of the parameters of the thrombin generation test between heathy subjects (control, n 5 20), patients with reactive thrombocytosis (RT, n 5 24) and patients with essential thrombocythemia (ET, n 5 29). Patients with RT had a longer lag time (A) than ET patients and controls. ETP (B), peak height thrombin generation curves (C) and velocity index of thrombin generation (D) were higher in RT patients than in ET patients whereas they were lower in ET patients than in controls. Data are presented as mean 6 SD. *P < 0.05 versus RT (Kruskal-Wallis rank sum test). TABLE II. Thrombin Generation Test Parameters and Results of Procoagulant Phospholipids Aassay in ET Patients According to JAK2 V617F Mutation Status and History of Thrombosis Lag time (min) ETP (nm 3 min) Peak (nm) Velocity index (nm/min) Procoagulant phospholipids (ratio a ) JAK2 V617F status Positive (n 520) Negative (n 5 8) History of thrombosis Yes (n 56) No (n 522) Figure 2. Results of the procoagulant microparticles assay in patients with reactive thrombocytosis (RT, n 5 16) and patients with essential thrombocythemia (ET, n 5 28) reported as ratio (tested sample clotting time/control plasma clotting time). The mean clotting time ratio was significantly lower in ET patients ( ) than in RT patients ( ). Results are expressed as mean 6 SD. *P < 0.05 versus RT (Kruskal-Wallis rank sum test). Data presented as mean (6SD). ET, essential thrombocytosis; ETP, endogenous thrombin potential. a Ratio was calculated as tested sample clotting time/control plasma clotting time. A shortening clotting time of sample compared with usual values of control plasma, expressed as a ratio <1, meant an increase in procoagulant phospholipids. history of thrombosis (Table II). Regarding procoagulant phospholipids, clotting time ratios were not significantly different between JAK2 V617F -positive and -negative ET patients (Table II). No significant differences were found between patients with and without history of thrombosis. Receiving operating curves (ROC) analysis and diagnostic values for ET The ROC curves were analyzed for each parameters of TGT and procoagulant phospholipids assay (Fig. 3). The lower bound of the AUC 95% confidence interval was greater than 0.7 for two of the parameters: lag time (AUC : 0.817; 95% CI ) and procoagulant phospholipids ratio (AUC : 0.853; 95% CI ) (Fig. 3). Individual results for each subject are presented in Figure 4 for lag time and Procoag-PPLVR ratio. A threshold was set for each of these two parameters allowing estimating a PPV, NPV, and a percentage of correctly classified. These thresholds were chosen in order to maximize the NPV and to maintain a satisfying PPV. In our study population, a lag time 3.3 min was associated with a PPV 5 70%, a NPV5 92.3% and a percentage of correctly classified % for ET. A procoagulant phospholipids ratio 0.76 was associated with a PPV 586.2%, a NPV % and a percentage of correctly classified % for ET. Discussion Thrombotic complications often occur in ET patients and are considered as their major cause of morbidity and mortality [6]. On the contrary, thrombotic events are infrequent in patients with RT [17]. Thus, identifying ET patients is essential in order to initiate an appropriate therapy in highrisk thrombocythemic patients prior to the occurrence of a vascular event and to avoid potentially harmful treatment in RT. However differentiating ET and RT is sometimes difficult and needs costly and/or invasive procedures. Despite the inclusion of JAK2 V617F mutation in the proposed criteria for ET, excluding RT remains crucial since this clonal marker is present only in about half of the ET patients [19]. American Journal of Hematology 1009

4 Figure 3. ROC curves illustrating the estimation of the diagnostic value for essential thrombocythemia of several parameters of the thrombin generation test and procoagulant phospholipids assay: lag time, endogenous thrombin potential (ETP), peak, velocity index, procoagulant phospholipids ratio. The area under the ROC curve (AUC) was (95% confidence interval [CI] ) for the lag time, (95% CI ) for the ETP, (95% CI ) for the peak height, (95% CI ) for the velocity index, and (95% CI ) for the procoagulant phospholipids ratio. Lag time and procoagulant phospholipids ratio were considered as relevant for the ET diagnosis since the lower bounds of their AUC 95% confidence interval were greater than 0.7. Figure 4. Individual values of lag-time (A) and procoagulant phospholipids (B) observed in patients with RT and ET. A lag-time 3.3 min or a Procoag-PPLVR ratio 0.76 were associated with optimal NPV for ET. In our study, we compared procoagulant potential between RT and ET patients, using two functional tests: thrombin generation test and procoagulant phospholipids assay. Compared with controls, ET patients had lower ETP and peak height values of thrombin generation. A similar decrease was reported in the studies of Marchetti et al. [20] and Duchemin et al. [16], in which a lower ETP was reported in ET patients [20] or MPN patients [16]. These lower ETP and peak height values did not persist in the presence of Activated Protein C [20]. In this condition, Marchetti et al. concluded that plasma from ET patient was APC-resistant which may account for or contribute to the prothrombotic state in myeloproliferative neoplasms [20]. In our study, despite a lower risk of developing thrombosis, RT patients presented higher ETP, peak height, and velocity values than ET patients, as determined by TGT parameters. It is noticeable that these TGT profiles in RT patients did not differ from controls, except for lag time. The prolonged lag time observed in RT patients could be related to a higher fibrinogen level associated with inflammatory and infectious states, frequently observed in RT patients and demonstrated in vitro by Duchemin et al. [21]. Similarly, prolonged reptilase times have been shown to be associated with elevated fibrinogen levels in acute phase reactions (i.e., inflammatory or infectious states) [22]. Indeed, and only in vitro, fibrinogen in an acute phase reaction may act dysfunctional (perhaps due to changes in fibrinogen s sialic acid or phosphorus content) [22] or interfere with the action of thrombin [21] on its substrate such that in the initiation phase its action could be slowed down and thrombin generation delayed. Thus the TGT profile observed in RT patients is in agreement with studies having determined that RT is not associated with a significant risk of thromboembolic events [17] unless additional risk factors are present. Besides, the measurement of procoagulant phospholipids showed a lower clotting time in ET and RT patients than in controls, and a significant reduction of the clotting time in ET patients compared with RT patients. This reflects a significant increase of procoagulant phospholipids concentration in ET patients, and to a lesser extent in RT patients. Several studies have already determined the concentration of circulating microparticles [15,18] or plasma microparticles 1010 American Journal of Hematology

5 procoagulant activity [16] in MPN patients. All these studies agreed that MPN patients had a higher concentration of circulating microparticles in plasma. Interestingly, Moles- Moreau et al. [18], using flow cytometry procedure, observed an increased ratio of platelet microparticles/platelet value in ET patients compared with controls or RT patients. Recent studies determined that the STA-Procoag-PPL clotting assay for microparticles activity was correlated with the platelets microparticles count obtained by flow cytometry [23,24] and by ELISA method [24]. It was shown that the increase of microparticles content and activity shortened the procoagulant clotting time of the STA-Procoag-PPL coagulation assay [24]. Evaluation of procoagulant phospholipids by this standardized coagulation assay could be of interest in prothrombotic states such as ET, considering that microparticles analysis by flow cytometry has still a lack of standardisation [25] and is only performed in specialized laboratories. Taken together, results of TGT and evaluation of procoagulant phospholipids support the hypothesis of the role of microparticles in the hypercoagulable state observed in ET. This confirms that mechanisms of thrombosis in ET patients imply cellular activation, such as platelets, leucocytes, and endothelial cells [26]. Concerning the JAK2 V617F mutation status and the results of TGT and procoagulant phospholipids in ET patients, no differences were found between patients with or without mutation. This is in line with the results of Panova-Noeva [27] with PPP or Trappenburg [15]. However, some authors found that patients with JAK2 V617F mutation (particularly homozygous) had a lower ETP than patients without mutation [16,20], and a higher circulating procoagulant activity [16]. In our study, no difference was observed between patients with or without history of thrombosis. These controversial data in literature can be explained by various elements: small sample sizes, differences in preanalytical procedures and in groups of studied patients (ET vs ET 1 polycythemia vera). The absence of significant differences in our study has to be interpreted with caution. Statistical analysis may lack power because of the small number of patients with history of thrombosis or because of the disproportion between patients with and without JAK2 V617F mutation. Differentiating ET and RT patients using rapid and available tests might be of interest. We therefore determined the diagnostic value of each parameter studied (lag time, ETP, peak height, velocity index, and clotting time ratio) by receiver operating characteristic analysis. Among these parameters, lag time, and ratio showed the best AUC value and the optimal negative predictive value. As limitation, these results were obtained in a small cohort. A study on an independent population would be the only way to get an unbiased estimation of sensitivity, specificity positive, and negative predictive values. Indeed, the positive and negative predictive values reported in this study should not be extrapolated to another population than the study sample. In conclusion, our study is the first one, to our knowledge, comparing a thrombin generation test and procoagulant phospholipids determination in ET and RT patients using standardized tests. These in vitro results imply that the mechanism of prothrombotic states in ET patients might relate to cellular activation and interaction. This potentially highlights the use of antiplatelet and/or cytoreduction therapy to prevent thrombotic events in myeloproliferative neoplasms. Finally, we demonstrated that these two standardized biological tests have the potential to further improve the differential diagnosis between ET and RT. Because our population is relatively small, these results should be validated in a larger cohort. References 1. Bleeker JS, Hogan WJ. Thrombocytosis: Diagnostic evaluation, thrombotic risk stratification, and risk-based management strategies. Thrombosis 2011;2011: Epub.2011 Jun Skoda RC. Thrombocytosis. Hematology Am Soc Hematol Educ Program 2009: James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005;434: Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005;365: Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med 2005;352: Landolfi R, Cipriani MC, Novarese L. Thrombosis and bleeding in polycythemia vera and essential thrombocythemia: Pathogenetic mechanisms and prevention. Best Pract Res Clin Haematol 2006;19: Elliott MA, Tefferi A. Thrombosis and haemorrhage in polycythaemia vera and essential thrombocythaemia. Br J Haematol 2005;128: Carobbio A, Finazzi G, Guerini V, et al. Leukocytosis is a risk factor for thrombosis in essential thrombocythemia: Interaction with treatment, standard risk factors, and Jak2 mutation status. Blood 2007;109: Wehmeier A, S udhoff T, Meierkord F. Relation of platelet abnormalities to thrombosis and hemorrhage in chronic myeloproliferative disorders. Semin Thromb Hemost 1997;23: Falanga A, Marchetti M, Evangelista V, et al. Polymorphonuclear leukocyte activation and hemostasis in patients with essential thrombocythemia and polycythemia vera. Blood 2000;96: Robertson B, Urquhart C, Ford I, et al. Platelet and coagulation activation markers in myeloproliferative diseases: Relationships with JAK2 V617F status, clonality, and antiphospholipid antibodies. J Thromb Haemost 2007;5: Arellano-Rodrigo E, Alvarez-Larran A, Reverter JC, et al. Increased platelet and leukocyte activation as contributing mechanisms for thrombosis in essential thrombocythemia and correlation with the JAK2 mutational status. Haematologica 2006;91: Falanga A, Marchetti M, Vignoli A, et al. Leukocyte-platelet interaction in patients with essential thrombocythemia and polycythemia vera. Exp Hematol 2005;33: Villmow T, Kemkes-Matthes B, Matzdorff AC. Markers of platelet activation and platelet-leukocyte interaction in patients with myeloproliferative syndromes. Thromb Res 2002;108: Trappenburg MC, van Schilfgaarde M, Marchetti M, et al. Elevated procoagulant microparticles expressing endothelial and platelet markers in essential thrombocythemia. Haematologica 2009;94: Duchemin J, Ugo V, Ianotto J-C, et al. Increased circulating procoagulant activity and thrombin generation in patients with myeloproliferative neoplasms. Thromb Res 2010;126: Griesshammer M, Bangerter M, Sauer T, et al. Aetiology and clinical significance of thrombocytosis: Analysis of 732 patients with an elevated platelet count. J Intern Med 1999;245: Moles-Moreau MP, Ternisien C, Tanguy-Schmidt A, et al. Flow cytometryevaluated platelet CD36 expression, reticulated platelets and platelet microparticles in essential thrombocythaemia and secondary thrombocytosis. Thromb Res 2010;126:e Tefferi A, Thiele J, Orazi A, et al. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelotibrosis: Recommendations from an ad hoc international expert panel. Blood 2007;110: Marchetti M, Castoldi E, Spronk HM, et al. Thrombin generation and activated protein C resistance in patients with essential thrombocythemia and polycythemia vera. Blood 2008;112: Duchemin J, Pan-Petesch B, Arnaud B, et al. Influence of coagulation factors and tissue factor concentration on the thrombin generation test in plasma. Thromb Haemost 2008;99: Van Cott EM, Smith EY, Galanakis DK. Elevated fibrinogen in an acute phase reaction prolongs the reptilase time but typically not the thrombin time. Am J Clin Pathol 2002;118: Gerotziafas GT, Van Dreden P, Chaari M, et al. The acceleration of the propagation phase of thrombin generation in patients with steady-state sickle cell disease is associated with circulating erythrocyte-derived microparticles. Thromb Haemost 2012; 107: Strasser EF, Happ S, Weiss DR, et al. Microparticle detection in platelet products by three different methods. Transfusion 2012;53: Lacroix R, Judicone C, Poncelet P, et al. Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol. J Thromb Haemost 2012;10: Cervantes F. Management of essential thrombocythemia. Hematology Am Soc Hematol Educ Program 2011;2011: Panova-Noeva M, Marchetti M, Spronk HM, et al. Platelet-induced thrombin generation by the calibrated automated thrombogram assay is increased in patients with essential thrombocythemia and polycythemia vera. Am J Hematol 2011;86: American Journal of Hematology 1011