| HOME | HELP | FEEDBACK | TABLE OF CONTENTS | ARCHIVE | SUBSCRIPTIONS |
|
|
|
||||||
|
||||||||
Myeloproliferative Disorders |
1 Laboratoire d’Hématologie, CHU, Bordeaux, France
2 Laboratoire d’Hématologie, CHU, Dijon, France
3 Murdoch University, Perth, Australia
4 University of Texas MD Anderson Cancer Center, Houston, Texas, USA
5 ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA
6 Uniklinik Eppendorf, Hamburg, Germany
7 Molecular Genetics, Royal Devon and Exeter Hospital, Exeter, UK
8 Molecular Diagnostics, Jeroen Bosch Hospital, s-Hertogenbosch, Netherlands
9 Laboratoire d’Hématologie, CHU, Clermont-Ferrand, France
10 Hematology Division, University of Utah School of Medicine, Salt Lake City, Utah, USA
11 Laboratoire d’Hématologie, CHU, Brest, France
12 Laboratoire de Cytologie Clinique et Cytogénétique, CHU, Nîmes, France
13 Laboratoire d'Hématologie, Hôtel-Dieu, Paris, France
14 Department of Hematology, University of Pavia and Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
15 Experimental Hematology, Department of Biomedicine, Basel University Hospital, Basel, Switzerland
16 Laboratoire d’Hématologie, CHU, Nantes, France
Correspondence: Sylvie Hermouet, Laboratoire d’Hématologie, Centre Hospitalier Universitaire, Institut de Biologie, 9 quai Moncousu, 44093 Nantes, France., E-mail:sylvie.hermouet{at}chu-nantes.fr
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.
Key words: JAK2V617F, quantification, standardization, allele-specific PCR, myeloproliferative diseases, multicenter study.
Related Article
Haematologica 2009 94: 7-10.
| HOME | HELP | FEEDBACK | TABLE OF CONTENTS | ARCHIVE | SUBSCRIPTIONS |
