Cell mediated cytotoxicity assessment by relative changes in viable target absolute counts.

We measured absolute counts (cells/microl) of Calcein-AM stained target cells that remained viable [i.e. not permeable to the viability probe 7-AAD (7-Amino Actinomycin D)] following a four-hour incubation with effectors [NK, CTL (cytotoxic T lymphocytes)] without using beads or standards. The absolute counts were evaluated by a Cytoron Absolute (Ortho) flow cytometry apparatus. Median triplicate counts were compared at 0 and 4h, in single targets and increased effector/target ratios. The Coefficient of Variation (CV) of the cell concentration (cells/microl) at the beginning of the experiment was below 6%. The % changes of viable target counts were correlated to the effector/target ratios by linear regression. The methodology was applied in pairs: 17 for allogenic stem cell transplantation from unrelated donors, 3 of healthy unrelated individuals, 10 for measuring NK activity and 7 autologous. In 15/20 allogenic MLC (mixed lymphocytes culture) and for all NK assays the cytotoxicity was positive (p < 0.05, r2 > 0.9) while in 5/20 allogeneic MLC and in all autologous MLC the outcome was negative (p > 0.05, r2 < 0.4). The proposed method offers the advantage of combining absolute counts with flow cytometry analysis of viable targets, assessment of cell behavior during the cytotoxic phenomenon, the use of a small amount of cells and excellent sensitivity. Our method can estimate frequencies higher than 1:100 for CTL assays.