The magnitude of qPCR reactivity prospectively identifies selected wells enriched for functionally active antigen specific T cells. Day 6 gp100209–217 sensitized microcultures (n = 24) from patients 1 (A.) and 3 (B.) were evaluated in parallel using qPCR and ELISA assays. Significant well reactivity was detected with the qPCR assay, but not with ELISA. Microcultures with the highest and lowest qPCR SIs were selected (see arrow) for rapid expansion. On day 14, the phenotype of the expanded cultures was assessed by staining with gp100209–217 peptide/HLA-A*0201 tetramers and anti-CD8 antibody and analysis by flow cytometric analysis. Dot plots are shown for propidium iodide–negative gated cells. Values in FACS dot plots correspond to the percentage of total CD8+ T cells that are tetramer-positive calculated as the number of CD8+ tetramer+ cells divided by the total number of CD8+ T cells minus the CD8- tetramer+ background × 100. Functional reactivity of the expanded cultures was assessed by co-culture with peptide pulsed T2 cells and supernatant analysis by standard ELISA for IFN-γ protein at 24 hrs. ELISA data represents the average of replicate co-culture wells. (O) represents the SI for each microwell. Shaded area represents range of non-specific reactivity (SI = 0.5–2.0). (*), not detectable.