Leukemia Research 35 (2011) 163–168

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Immunophenotype of acute myeloid leukemia with NPM mutations: Prognostic impact of the leukemic compartment size
J. Nomdedeu a,∗ , E. Bussaglia a , N. Villamor b , C. Martinez a , J. Esteve b , M. Tormo c , C. Estivill a , M.P. Queipo d , R. Guardia e , M. Carricondo a , M. Hoyos a , A. Llorente f , J. Juncà g , M. Gallart h , A. Domingo i , J. Bargay j , M. Mascaró j , J.M. Moraleda k , L. Florensa l , J.M. Ribera g , D. Gallardo e , S. Brunet a , A. Aventin a , J. Sierra a , on behalf of the Spanish CETLAM a Department of Hematology and Laboratory, Hospital de la Santa Creu I …ver más…

Quiescent leukemic stem cells (LSC) would be responsible of the maintenance of all the leukemic lineages, their persistence after treatments and clinical relapses [1,2]. Flow cytometry provides a useful tool to characterize heterogeneous populations such as those encountered in human leukemias. Mutations in the nucleophosmin gene are the most commonly

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J. Nomdedeu et al. / Leukemia Research 35 (2011) 163–168 optimized for retrotranscription. In all, 1 g of total RNA was retrotranscribed in a total reaction volumen of 20 l containning Cl2 Mg 5 mM, 10× buffer, DTT 10 mM, dNTP’s 10 mM each, random hexamers 15 M, RNAsin 20 units and 200 units of RT enzyme. Samples were incubated for 10 mim at 20 ◦ C, 45 min at 42 ◦ C and 3 min at 99 ◦ C followed by 10 min at 4 ◦ C. Quantitative NPM type A transcript level was determined by RT-PCR at diagnosis and at follow up using ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA). The PCR reaction was carried out in a 25 l reation with 5 l of cDNA, 12,5 l (2×) Taqman Universal PCR Master Mix, 300 nM of primers and 200 nM concentration NPM and ABL probes [16,17]. The reaction conditions were 50 ◦ C for 2 min and 95 ◦ C for 10 min, followed by 50 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min for annealing and extension. All PCR experiments were performed in triplicate. Standard curves for NPM and ABL were obtained using dilution series of plasmids (Ipsogen,