Interphase fluorescence in situ hybridization (I-FISH) studies have a remarkable prognostic value in patients with chronic lymphocytic leukemia (CLL). I-FISH studies can be performed either on tetradecanoylphorbol acetate stimulated peripheral blood cells (I-FISH-TPA) or unstimulated peripheral blood mononuclear cells (I-FISH-PBMC). The aim of the study was to evaluate whether this finding was clinically relevant in a group of 235 patients with CLL. Fifty-six patients had both I-FISH-TPA and I-FISH-PBMC results. Compared with uncultured cells, the cytogenetic detection rate rose from 57 to 80% with the use of TPA-stimulated cells (P = 0.014). I-FISH-TPA provided a better prediction of treatment-free survival compared with I-FISH-PBMC (P = 0.031 vs. 0.166). Then, I-FISH-PBMC results from 93 historical patients were compared with 86 recent patients with I-FISH-TPA results. Genomic aberrations were detected in 46 and 67% of patients from the I-FISH-PBMC and I-FISH-TPA cohorts, respectively. The detection rate of 13q deletion as the only aberration increased from 10% with I-FISH-PBMC to 37% with I-FISH-TPA (P = 0.006). In conclusion, I-FISH-TPA increased the detection rate of 13q deletion and had an improved prognostic value compared with I-FISH-PBMC.