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“The JAK2 V617F mutation is detectable in granulocyte populations at greater than two copies per cell among individuals with myeloproliferative disorders”, by El Housni et al.

Leukemia volume 21, pages 817–818 (2007)Cite this article

In our recent letter to Leukemia,1 we reported that in lieu of a definitive negative control, or the availability of an alternative ‘gold standard’ methodology to verify the accuracy of our quantitative assay for the JAK2 V617F mutation, we have used healthy controls as a surrogate to assess assay specificity, and also to establish a clinically useful range for supporting a diagnosis of myeloproliferative disorder.

We consider that amplification of small numbers of mutant JAK2 copies in normals could represent either mis-priming, or the presence of 3′-shortened mutation-specific primers (as it is only the very 3′-end base that confers specificity, and 3′-shortened primers will amplify from both wild type and mutant DNA). The primers in our assay were high-performance liquid chromatography (HPLC)-purified and we avoided freeze thawing. However, any reductions in the integrity and quality of the oligonucleotides could potentially contribute to non-specific amplification. Our assumption that low-level amplification of the JAK2 V617F sequence in healthy controls may represent artefact of the polymerase chain reaction (PCR) reaction, is also based on a previous publication,2 where the authors demonstrated that the specificity of their assay to quantify donor and recipient hemopoietic cells by realtime–PCR of single nucleotide polymorphisms required a cutoff level for the crossing threshold (Ct) of 35 to exclude amplification of the negative allele. Similarly, we did not know whether amplification in healthy controls and in lymphocyte DNA after 36 cycles in our assay represented non-specific amplification of the wild-type JAK2 allele (or granulocyte contamination in the case of DNA from the lymphocyte fraction).

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References

  1. Hammond E, Shaw K, Herrmann R . The JAK2V617F mutation is detectable in granulocyte populations at greater than 2 copies per cell among individuals with myeloproliferative disorders. Leukemia, published online.

  2. Maas F, Schaap N, Kolen S, Zoetbrood A, Bun I, Dolstra H, de Witte T et al. Quantification of donor and recipient hemopoietic cells by real-time PCR of single nucleotide polymorphisms. Leukemia 2003; 17: 630–633.

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  3. Hammond E, Shaw K, Carnley B, P'ng S, James I, Herrmann R . Quantitative determination of JAK2 V617F by TaqMan: an absolute measure of averaged copies per cell that may be associated with the different types of myeloproliferative disorders. J of Mol Diagnostics, (in press).

  4. Sidon P, El Housni H, Dessars B, Heimann P . The JAK2V617F mutation is detectable at very low level in peripheral blood of healthy donors. Leukemia, 15 June 2006, published online.

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Authors and Affiliations

  1. Centre for Clinical Immunology and Biomedical Statistics, Murdoch University, Western Australia, Australia

    E Hammond

  2. Cellular and Tissue Therapies, Royal Perth Hospital, Western Australia, Australia

    K Shaw

  3. Department of Haematology, Pathwest, Royal Perth Hospital, Western Australia, Australia

    R Herrmann

Authors
  1. E Hammond
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  2. K Shaw
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  3. R Herrmann
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Correspondence to E Hammond.

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Hammond, E., Shaw, K. & Herrmann, R. “The JAK2 V617F mutation is detectable in granulocyte populations at greater than two copies per cell among individuals with myeloproliferative disorders”, by El Housni et al.. Leukemia 21, 817–818 (2007). https://doi.org/10.1038/sj.leu.2404569

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  • DOI: https://doi.org/10.1038/sj.leu.2404569

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