Concordance of assays designed for the quantification of JAK2V617F: a multicenter study.
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.
Lippert
Eric
E
Laboratoire d'Hématologie, Centre Hospitalier Universitaire, Institut de Biologie, Bordeaux, France.
Girodon
François
F
Hammond
Emma
E
Jelinek
Jaroslav
J
Reading
N Scott
NS
Fehse
Boris
B
Hanlon
Katy
K
Hermans
Mirjam
M
Richard
Céline
C
Swierczek
Sabina
S
Ugo
Valérie
V
Carillo
Serge
S
Harrivel
Véronique
V
Marzac
Christophe
C
Pietra
Daniela
D
Sobas
Marta
M
Mounier
Morgane
M
Migeon
Marina
M
Ellard
Sian
S
Kröger
Nicolaus
N
Herrmann
Richard
R
Prchal
Josef T
JT
Skoda
Radek C
RC
Hermouet
Sylvie
S
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
D010649:Phenylalanine; D004247:DNA; D053614:Janus Kinase 2; D014633:Valine