Concordance of assays designed for the quantification of JAK2V617F: A multicenter study

Eric Lippert, François Girodon, Emma Hammond, Jaroslav Jelinek, N. Scott Reading, Boris Fehse, Katy Hanlon, Mirjam Hermans, Céline Richard, Sabina Swierczek, Valérie Ugo, Serge Carillo, Véronique Harrivel, Christophe Marzac, Daniela Pietra, Marta Sobas, Morgane Mounier, Marina Migeon, Sian Ellard, Nicolaus KrögerRichard Herrmann, Josef T. Prchal, Radek C. Skoda, Sylvie Hermouet

Research output: Contribution to journalArticle

Abstract

Background: Many different techniques have been designed for the quantification of JAK2V617F allelic burden, sometimes producing discrepant results. Design and Methods: JAK2V617F quantification techniques were compared among 16 centers using 11 assays based on quantitative polymerase chain reaction (with mutation-specific primers or probes, or fluorescent resonance energy transfer/melting curve analysis), allele-specific polymerase chain reaction, conventional sequencing or pyrosequencing. Results: A first series of blinded samples (granulocyte DNA, n=29) was analyzed. Seven assays (12 centers) reported values inside the mean±2SD; the mean coefficient of variation was 31%. Sequencing techniques lacked sensitivity, and strong discrepancies were observed with four techniques, which could be attributed to inadequate standards or to different modes of expression of results. Indeed, quantification of JAK2V617F in relation to another control gene produced higher than expected values, suggesting the possibility of more than two JAK2 copies/cell. After calibration of assays with common 1% to 100% JAK2V617F standards (dilutions of UKE-1 cells in normal leukocytes), 14 centers tested ten new samples. JAK2V617F allelic burdens greater or equal than 1% were then reliably quantified by five techniques - one allele specific-polymerase chain reaction and four TaqMan allele-specific quantitative polymerase chain reaction assays, including one previously giving results outside the mean±2SD - with a lower mean coefficient of variation (21%). Of these, only the two TaqMan allele-specific quantitative polymerase chain reaction assays with primerbased specificity could detect 0.2% JAK2V617F. Conclusions: Techniques expressing the allelic burden as JAK2V617F/total JAK2 and using a common set of standards produced similar quantification results but with variable sensitivity. Calibration to a reference standard improved reproducibility.

Original languageEnglish
Pages (from-to)38-45
Number of pages8
JournalHaematologica
Volume94
Issue number1
DOIs
Publication statusPublished - Jan 2009

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Keywords

  • Allele-specific PCR
  • JAK2V617F
  • Multicenter stud
  • Myeloproliferative diseases
  • Quantification
  • Standardization

ASJC Scopus subject areas

  • Hematology

Cite this

Lippert, E., Girodon, F., Hammond, E., Jelinek, J., Reading, N. S., Fehse, B., Hanlon, K., Hermans, M., Richard, C., Swierczek, S., Ugo, V., Carillo, S., Harrivel, V., Marzac, C., Pietra, D., Sobas, M., Mounier, M., Migeon, M., Ellard, S., ... Hermouet, S. (2009). Concordance of assays designed for the quantification of JAK2V617F: A multicenter study. Haematologica, 94(1), 38-45. https://doi.org/10.3324/haematol.13486