A higher JAK2 V617F-mutated clone is observed in platelets than in granulocytes from essential thrombocythemia patients, but not in patients with polycythemia vera and primary myelofibrosis Leukemia (2007) 21, 1331–1332. doi:10.1038/sj.leu.2404649; published online 15 March 2007 Essential thrombocythemia (ET) is a chronic myeloproliferative disorder characterized by megakaryocyte hyperplasia and thrombocytosis. The JAK2 V617F mutation has been reported in a variable proportion of ET patients ranging from 23 to 57%. These percentages have been determined in the majority of studies using granulocytes as the nucleic acid source and depend on the sensitivity of the detection techniques applied, being direct sequencing and allele-specific (AS)-PCR the most frequently used (reviewed by Steensma et al. 1 ). Application of more sensitive techniques such as quantitative real time PCR in DNA from granulocytes has yielded even higher detection rates (75%) mainly in newly diagnosed ET patients. 2 Although ET is characterized by a predominant mega- karyocytic-platelet lineage involvement, there is scarce in- formation about the prevalence of JAK2 V617F mutation in platelets. We have described the presence of the V617F mutation in growth factor-independent megakaryocytic colonies and also in platelets from five out of six ET patients. 3 In a recent study, JAK2 V617F was reported in 24 out of 50 ET patients when platelets were analyzed by AS-RT-PCR and restriction fragment length polymorphism. 4 Comparison of JAK2 V617F mutational status by conventional AS-PCR in granulocytes and platelets from 10 ET patients showed no differences in the detection rate between the two populations studied. 5 More recently, quantification of the JAK2 V617F in platelets and granulocytes in 13 ET patients showed a higher allele percentage in platelets than in granulocytes in a study that examined the reciprocity between JAK2 mutational status and Mpl expression. 6 Our aim was to systematically apply quantitative AS-RT-PCR to evaluate and compare the prevalence of the JAK2 V617F mutation in granulocytes and platelets in a series of 75 ET patients from a single institution. ET patients were diagnosed according to the WHO criteria. At the time when JAK2 mutation was analyzed 24/75 patients were receiving platelet-lowering therapy 7ASA, 24/75 patients only received antiaggregants and 27/75 received no specific treatment. None of them was receiving interferon. Twenty milliliter of venous blood was collected in ethylenediaminetetraacetic acid and immediately processed. Platelet-rich plasma was obtained by centrifugation of anticoagulated whole blood at 194 g for 10 min. Granulocytes were isolated by Lymphoprep (1077 g/ml) density gradient, followed by dextran sedimentation. Total RNA was isolated from granulocytes or platelets using guanidinium thiocyanate method (Ultraspec; Biotecx Laboratories, Houston, TX, USA). cDNA was reverse transcribed from 1 mg of total RNA with Murine Moloney Leukemia Virus reverse transcriptase (Invitro- gen, Life Technologies, CA, USA) according to standard procedures with random hexamers. JAK2 V617F mutation was analyzed in duplicate by real-time AS-RT-PCR with probes specific for the mutated and the wild-type form as described previously. 7 A ratio between the Ct JAK2V617F and Ct JAK2WT was calculated for each sample. The JAK2 V617F mutation was detected in both granulocytes and platelets in 43 out of 75 ET patients (57%), and was negative in both cell populations in the remaining 32 patients. It is worth to mention that the JAK2 V617F mutation had not been detected in six of the 43 mutated cases by direct sequencing using granulocyte cDNA or conventional AS-PCR performed with granulocyte DNA (following the methodology described by Baxter et al. 8 ). Although no difference was observed in the overall detection rate, when comparing platelets and granulocytes in the JAK2 V617F-positive cases, the mean Ct(JAK2V617F)/Ct(JAK2 wild- type) ratio was significantly higher, 1.05770.067, for granulo- cytes than for platelets, 1.02170.042 (P ¼ 0.003) (Figure 1a). These results show that higher C t values for the mutated form were obtained when granulocytes were analyzed and reflect that the amount of the mutated form of JAK2 is lower in granulocytes than in platelets. To ascertain whether this 1.2 1.1 1.0 0.9 N = 43 43 50 40 30 20 10 0 0 10 20 Granulocyte V617FJAK2 (%) 30 40 50 Granulocytes Platelets p = 0.003 Platelet V617FJAK2 (%) Ratio Ct(V617F JAK2)/Ct(wild type JAK2) b a Figure 1 Quantification of JAK2V617F allelic burden in granulo- cytes and platelets from ET patients. (a) Representation of the C t ratio in granulocytes and platelets of the 43 mutated cases. (b) Distribution of the allelic burden observed in granulocytes and platelets from the 43 JAK2 V617F-positive ET patients. Letters to the Editor 1331 Leukemia