Increased circulating procoagulant activity and thrombin generation in patients with myeloproliferative neoplasms.

Microparticles (MPs) are membrane fragments ranging in size from 0.1 to 1 microm, and are considered as biomarkers reflecting prothrombotic state in many clinical diseases. The clinical course of myeloproliferative neoplasms (MPN) being frequently complicated by thrombotic events, we determined the MPs activity, i.e. circulating procoagulant activity (CPA), in polycythemia vera (PV) and essential thrombocythemia (ET) patients. To evaluate the influence of MPs on the coagulation, a thrombin generation test was realized in the absence and presence of thrombomodulin (TM). Compared with controls, patients had a higher CPA (24.0+/-9.0 vs 10.6+/-4.4 nM, p<0.001), which was associated with a lower inhibition of the thrombin generation in the presence of TM (20.1+/-9.5% vs 28.4+/-11.8%, p<0.001), compatible with a low sensitivity to TM. This sensitivity was influenced by the JAK2V617F mutational status, homozygous patients presenting the lowest inhibition rate of the thrombin generation. Filtration through a 0.22 microm membrane increased the sensitivity to TM in plasma, suggesting an influence of MPs in the "TM-resistance" observed in patients. Moreover, MPN patients receiving antiplatelet and/or cytoreductive therapies, our study suggests that CPA might be influenced by cytoreductive therapy. In conclusion, our data evidence in MPN patients the occurrence of an acquired "TM-resistance" partly determined by circulating microparticles. This TM-resistance might contribute to the hypercoagulable state observed in MPN patients, but the predictive value of the "TM-resistance" for thrombosis had not been evaluated.

Interlaboratory development and validation of a HRM method applied to the detection of JAK2 exon 12 mutations in polycythemia vera patients.

BACKGROUND: Myeloproliferative disorders are characterized by clonal expansion of normal mature blood cells. Acquired mutations giving rise to constitutive activation of the JAK2 tyrosine kinase has been shown to be present in the majority of patients. Since the demonstration that the V617F mutation in the exon 14 of the JAK2 gene is present in about 90% of patients with Polycythemia Vera (PV), the detection of this mutation has become a key tool for the diagnosis of these patients. More recently, additional mutations in the exon 12 of the JAK2 gene have been described in 5 to 10% of the patients with erythrocytosis. According to the updated WHO criteria the presence of these mutations should be looked for in PV patients with no JAK2 V617F mutation. Reliable and accurate methods dedicated to the detection of these highly variable mutations are therefore necessary. METHODS/FINDINGS: For these reasons we have defined the conditions of a High Resolution DNA Melting curve analysis (HRM) method able to detect JAK2 exon 12 mutations. After having validated that the method was able to detect mutated patients, we have verified that it gave reproducible results in repeated experiments, on DNA extracted from either total blood or purified granulocytes. This HRM assay was further validated using 8 samples bearing different mutant sequences in 4 different laboratories, on 3 different instruments. CONCLUSION: The assay we have developed is thus a valid method, adapted to routine detection of JAK2 exon 12 mutations with highly reproducible results.