Estimation of telomerase activity by TRAP. (A) Representative profiles of TRAP assay performed using B-cell lysates prepared from 4 B-CLL patients. Each lysate was tested before (+) and after (Δ) heat inactivation. Samples 1, 2, and 3 expressed various levels of telomerase, whereas sample 4 lacked telomerase activity. A positive control assay was performed with each assay. (B) Telomerase activity was quantified in purified B-cell populations from 60 B-CLL patients and 24 aging healthy donors. Telomerase activity of B-CLL cells grouped as a whole was not different than that detected in B cells from healthy subjects. Unmutated cells from B-CLL patients expressed significantly elevated telomerase activity compared with mutated cells from B-CLL patients (P < .001). Telomerase activity of unmutated B cells from B-CLL patients did not differ significantly from that observed in the group of healthy donors. When compared independently, the mutated cells displayed significantly lower telomerase activity than B cells from healthy donors (P < .001). (C) Telomerase activity of flow-sorted B-cell subsets (CD19⁺CD5⁺ and CD19⁺CD5^–) from 8 aging healthy subjects compared with that from B cells of unmutated and mutated B-CLL (n = 30 each). Mutated B-CLL exhibited significantly lower telomerase activity than unmutated B-CLL (P < .001) and normal B-cell subsets (P < .01). (D) Scatter plot of mean telomere length compared with telomerase in entire cohort of B-CLL patients. There was a significant indirect correlation between mean telomere length and telomerase in the entire cohort of B-CLL patients (P = .043); however, the correlation of these 2 parameters did not reach statistical significance when the B-CLL patients were analyzed as individual subgroups (P = .067 for unmutated and P = .079 for mutated).