title: Concordance of assays designed for the quantification of JAK2V617F: a multicenter study.
abstract: BACKGROUND
  Many different techniques have been designed for the quantification of JAK2V617F allelic burden, sometimes producing discrepant results.
  
  
  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.
authors.affiliation: Laboratoire d'Hématologie, Centre Hospitalier Universitaire, Institut de Biologie, Bordeaux, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Lippert
authors.forename: François
authors.initials: F
authors.lastname: Girodon
authors.forename: Emma
authors.initials: E
authors.lastname: Hammond
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Jelinek
authors.forename: N Scott
authors.initials: NS
authors.lastname: Reading
authors.forename: Boris
authors.initials: B
authors.lastname: Fehse
authors.forename: Katy
authors.initials: K
authors.lastname: Hanlon
authors.forename: Mirjam
authors.initials: M
authors.lastname: Hermans
authors.forename: Céline
authors.initials: C
authors.lastname: Richard
authors.forename: Sabina
authors.initials: S
authors.lastname: Swierczek
authors.forename: Valérie
authors.initials: V
authors.lastname: Ugo
authors.forename: Serge
authors.initials: S
authors.lastname: Carillo
authors.forename: Véronique
authors.initials: V
authors.lastname: Harrivel
authors.forename: Christophe
authors.initials: C
authors.lastname: Marzac
authors.forename: Daniela
authors.initials: D
authors.lastname: Pietra
authors.forename: Marta
authors.initials: M
authors.lastname: Sobas
authors.forename: Morgane
authors.initials: M
authors.lastname: Mounier
authors.forename: Marina
authors.initials: M
authors.lastname: Migeon
authors.forename: Sian
authors.initials: S
authors.lastname: Ellard
authors.forename: Nicolaus
authors.initials: N
authors.lastname: Kröger
authors.forename: Richard
authors.initials: R
authors.lastname: Herrmann
authors.forename: Josef T
authors.initials: JT
authors.lastname: Prchal
authors.forename: Radek C
authors.initials: RC
authors.lastname: Skoda
authors.forename: Sylvie
authors.initials: S
authors.lastname: Hermouet
mesh_terms: D002138:Calibration; D002460:Cell Line; D004247:DNA / Q000235:genetics; D006801:Humans; D053614:Janus Kinase 2 / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D009196:Myeloproliferative Disorders / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D014633:Valine / Q000235:genetics / Q000378:metabolism
chemical_list: D010649:Phenylalanine; D004247:DNA; D053614:Janus Kinase 2; D014633:Valine