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

Lippert, Eric and Girodon, François and Hammond, Emma and Jelinek, Jaroslav and Reading, N. Scott and Fehse, Boris and Hanlon, Katy and Hermans, Mirjam and Richard, Céline and Swierczek, Sabina and Ugo, Valérie and Carillo, Serge and Harrivel, Véronique and Marzac, Christophe and Pietra, Daniela and Sobas, Marta and Mounier, Morgane and Migeon, Marina and Ellard, Sian and Kröger, Nicolaus and Herrmann, Richard and Prchal, Josef T. and Skoda, Radek C. and Hermouet, Sylvie. (2009) Concordance of assays designed for the quantification of JAK2V617F : a multicenter study. Haematologica : journal of hematology, Vol. 94. S. 38-45.

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Official URL: http://edoc.unibas.ch/dok/A6006886

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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 primer-based 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.
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel > Experimental Hematology (Skoda)
03 Faculty of Medicine > Bereich Medizinische Fächer (Klinik) > Ehemalige Einheiten Medizinische Fächer (Klinik) > Onkologie (Herrmann)
03 Faculty of Medicine > Departement Klinische Forschung > Bereich Medizinische Fächer (Klinik) > Ehemalige Einheiten Medizinische Fächer (Klinik) > Onkologie (Herrmann)
UniBasel Contributors:Skoda, Radek C. and Herrmann, Richard
Item Type:Article, refereed
Bibsysno:Link to catalogue
ISSN:0017-6567
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:24 May 2013 09:21
Deposited On:24 May 2013 08:59

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