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Comparison of whole blood vs purified blood granulocytes for the detection and quantitation of JAK2V617F

Leukemia volume 21, pages 1128–1130 (2007)Cite this article

The V617F mutation of JAK2 (JAK2V617F) is specific of myeloproliferative disorders (MPDs) and its detection is now one of the first tests performed in the diagnostic work-up of polycythemia and thrombocytoses.1, 2, 3 The JAK2V617F allele burden varies depending on the type of MPD, with the majority of polycythemia vera (PV) expressing >50% JAK2V617F and essential thrombocythemia (ET) expressing <50% mutant4, 5 in purified granulocytes, the source of DNA typically used to study the JAK2V617F allelic ratio. However, granulocyte purification being time-consuming, many diagnostic laboratories opt to work on whole blood instead. There are potential problems with using DNA extracted from whole blood rather than purified granulocytes. B and T lymphocytes typically do not express JAK2V617F, which implies that the JAK2V617F burden is likely to be underestimated in whole blood assays. Moreover, some PV and half of JAK2-mutated ET express <15% JAK2V617F: such low levels of mutant, often missed by sequencing, are detected in purified granulocytes by sensitive allele-specific PCR (AS-PCR) techniques. In whole blood assays however, some of the patients with low levels of mutant might be found negative.

We used a allele-specific, quantitative PCR (AS-qPCR) that detects 0.1% JAK2V617F in granulocytes5, 6 to compare results obtained with genomic DNA extracted from whole blood or purified blood granulocytes. Forty-eight consecutive patients, 41 with MPDs (12 PV, 26 ET and three idiopathic myelofibrosis (IMF)) and seven with secondary erythrocytosis (SE), were examined. Twelve MPD patients (five PV, five ET and two IMF) had been receiving cytoreductive treatment (pipobroman or hydroxyurea); all other patients were examined at the time of diagnosis. Aliquots of whole blood and pellets of purified granulocytes were prepared from the same sample, as described5, 7 and stored at −20°C (whole blood) or −80°C (granulocyte pellets) until use. Whole blood DNA and granulocyte DNA were prepared using QiaAmp DNA mini-kits (Qiagen) following the manufacturers' instructions. JAK2 AS-qPCRs were performed at least in duplicate with 25 ng DNA per reaction, as described previously;5, 6 copy numbers were determined by comparison with serial dilutions of plasmids containing wild-type and V617F JAK2 DNA amplicons.

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Authors and Affiliations

  1. Laboratoire d'Hématologie, Centre Hospitalier Universitaire de Nantes, Nantes, France

    S Hermouet, M-C Boursier, L Ergand, F Perrault-Hu & D Pineau

  2. Laboratoire d'Hématologie, Centre Hospitalier Universitaire d'Angers, Angers, France

    I Dobo

  3. Laboratoire d'Hématologie, Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France

    E Lippert

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  1. S Hermouet
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Correspondence to S Hermouet.

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Hermouet, S., Dobo, I., Lippert, E. et al. Comparison of whole blood vs purified blood granulocytes for the detection and quantitation of JAK2V617F. Leukemia 21, 1128–1130 (2007). https://doi.org/10.1038/sj.leu.2404588

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