CD39⁺/CD73⁺ CLL cells generate ADO from ADP. An HPLC assay was adopted to follow the enzymatic conversion of ADP to AMP (CD39 dependent) and to ADO (CD73 dependent). (A) Representative CD39⁺/CD73⁺ (left) and CD39⁺/CD73⁻ (right) patients are shown at t = 0 and after 60 minutes of incubation with 100μM ADP under physiologic conditions. ADP, AMP, and ADO are shown in a single HPLC run. Cumulative data of ADO production in presence or absence of 10μM APCP (B), ADP consumption (C), or AMP production (D) in CD73⁻ and CD73⁺ CLL patients. A functional cut-off for CD73 expression was designated at 30%. Peak identities were confirmed by comparing the retention times and the UV spectra of samples with standard compounds. Concentrations were calculated by comparing the peak area of samples with calibration curves for peak areas of each standard. All concentrations are expressed as micromoles per hour per 10⁶ cells. Statistical analysis was performed using Mann-Whitney U test. n = 20.