Leukoreduction system (LRS) chambers from the Trima Accel apheresis platform are widely used as a convenient source of human peripheral blood mononuclear cells (PBMCs) for immunological research. However, increasing demand has prompted the search for alternative sources. Here, we investigated whether residual cell concentrates recovered from Trima Accel tubing sets, typically discarded after platelet apheresis, could serve as a substitute for concentrates obtained from LRS chambers. Paired samples from 20 healthy platelet donors were analyzed for recovered volume, cellular composition and yield, leukocyte subset distribution, and functional responsiveness. Tubing sets contained significantly greater volumes (23.04 ± 14.1 ml vs. 8.14 ± 0.97 ml) but lower leukocyte concentrations (35.19 ± 16.99 × 10³/µl vs. 147.60 ± 59.63 × 10³/µl) than LRS chambers. Absolute leukocyte numbers were slightly higher in LRS chambers. However, cell frequencies in tubing sets showed a minor enrichment of monocytes and a modest lymphocyte depletion. Detailed flow cytometry characterization further revealed comparable distributions of major leukocyte populations and T cell subsets, including naïve and memory compartments, without biologically meaningful differences in activation or differentiation markers. Importantly, PBMCs from both sources exhibited comparable functional responses following phytohemagglutinin (PHA) stimulation, including similar changes in activation marker expression and cytokine production. Short-term storage of tubing set samples for 24 h at 4 °C did not compromise cell viability. Overall, tubing sets represent a functionally equivalent and practical alternative leukocyte source for research applications.