Book of abstract 2008

Comparison of the results for the JAK2 V617F mutation detection by
three methods; allele specific PCR and two real-time quantitative PCR
on polycythemia vera and essential thrombocythemia DNA samples
Tadej Pajič1, Eva Kralj1, Joško Vučković1, Peter Černelč1
1Dept. of Haematology, Division of Internal Medicine, University Clinical Centre Ljubljana, Zaloška 7,
SI-1000 Ljubljana, Slovenia; 2Dept. of Haematology, Division of Internal Medicine, General Hospital
Celje, Oblakova 5, SI-3000 Celje, Slovenia
JAK2 V617F is a clonal acquired mutation found in the majority of patients
with polycythemia vera (PV), and a significant number of patients with essential
thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF). The incidence
of this mutation ranging from 65% to 97% in PRV patients, from 35 % to 70 % in ET
patients and about 50 % in MMM depending on the study. These variations in percentage
of patients involved is likely due to the criteria used for diagnosis and also the sensitivity of
the assay used to detect this mutation. Allele specific PCR and different quantitative realtime
PCR (RQ-PCR) based methods are usable techniques for its detection.
The aim of this study was to compare the results for the JAK2 V617F mutation detection
on DNK of granulocytes of the PV and ET patients by three frequentlly used techniques.
The results were compared to that published in the literature.
The diagnosis of ET and PV was established following World Health Organization
classification. 19 patients (median age 69 years) with PV and 70 patients (median age 56
years) with ET were inculded in the study. EDTA peripheral blood was drawn and used
for the isolation of the granulocytes after ficoll density centrifugation. DNA was isolated
from granulocytes by QIAamp® DNA Mini Kit from Qiagen. The allele specific PCR was
carried out as described in Baxter EJ et al.(Lancet 2005; 365:1054-1061). RQ-PCR were
carried out by JAK2 MutaScreen Kit and JAK2 MutaQuant Kit (both Ipsogen).
Two
p22
patients (1 ET, 1 PV) that were negative by qualitative allele-specific PCR, were
proven to be positive for the JAK2 V67F mutation by both RQ-PCR methods. However,
there was also a PV patient that was negative by JAK2 MutaScreen Kit, but was positive
by other two methods. The percentage of positivity for JAK2 V617F mutation in ET
and PV patients were 63 % (44/70) and 100 % (19/19), respectively. The results were
calculated by JAK2 MutaQuant Kit.
We obtained the best sensitivity for detection of JAK2 V617F mutation on DNA samples
from granulocytes from peripheral blood by JAK2 MutaQuant Kit. The negative
detection of JAK2 V617F by JAK2 MutaScreen Kit in PV patient that has been proven
to be positive for the mutation by other two methods must be studied further. The
percentage of positivity for JAK2 V617F mutation in ET and PV patients was similar to
that published in the literature and was in the upper part of the range.
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