Detection of JAK2 V617F as a first intention diagnostic test for erythrocytosis.

The diagnosis of polycythemia vera (PV) is currently based on clinical and biological criteria defined either by the World Health Organization (WHO) or the Polycythemia Vera Study Group (PVSG).1, 2 Both of these classifications use clinical and biological markers organized in major and minor criteria, allowing to diagnose a PV when a define combination of major and minor criteria is present. Recently, a somatic point mutation of the tyrosine kinase JAK2 (JAK2 V617F) has been described in about 80% of PV patients as well as 30% of essential thrombocythemia (ET) and 50% of idiopathic myelofibrosis (IMF)3, 4, 5, 6 and several in vitro and in vivo experiments demonstrated that this mutation of JAK2 was the molecular event at the origin of PV.3 It was therefore tempting to use the detection of JAK2V617F as a diagnostic test for erythrocytosis. Such an attitude supposes the development of other techniques than sequencing, as this latter is time consuming and not always feasible in hematology laboratories. We thus compared sequencing with two techniques of real-time PCR-based mutation detection (one using the LightCycler® instrument, the other the Taqman® ABI Prism 7500), for the efficiency to detect the JAK2 V617F mutation in 119 samples from patients with suspicion of myeloproliferative disorder (MPD). The three techniques were equivalent in all samples but one, where sequencing failed to detect the mutation revealed by both LightCycler® and Taqman® technologies. To evaluate the sensitivity of these techniques, we tested serial dilutions of the homozygously mutated HEL cell line DNA in the nonmutated TF-1 cell line DNA, and serial dilutions of the genomic DNA from a patient homozygous for the JAK2 V617F mutation in normal DNA. Sequencing failed to detect the mutated allele under 5% of HEL cell line DNA diluted in TF-1 cell line DNA, and under 10% of the homozygously mutated patient's DNA diluted in normal DNA. The sensitivity of LightCycler® and Taqman® techniques were equivalent, slightly higher than sequencing, reaching 0.5–1% of HEL cell line DNA diluted in TF-1 cell line DNA (Figure 1), and 2–4% of a homozygously mutated patient's DNA diluted in normal DNA.