HPC viability measurement: trypan blue versus acridine orange and propidium iodide

BACKGROUND: A reliable, validated method for rapidly determining HPC viabil ity is essential for clinical cell engineering. STUDY DESIGN AND METHODS: A fluorometric cell viability assay using acridin e orange and propidium iodide (AO/PI) was compared to the current standard, trypan blue (TB) exclusion. Viable cells stained with AO/PI fluoresce gree n under darkfield fluorescence microscopy, while nonviable cells fluoresce orange. Mixtures of fresh and heat-killed bone marrow were prepared and use d as viability standards for evaluation of both assays. The frequency of CF U-GM was determined for each specimen. RESULTS: Cell viability measured by AO/PI was extremely linear, with measur ed and predicted viability in agreement from 0 to 100 percent of the viable cells and a coefficient of regression (r(2)) Of 0.9921. The predicted-viab ility regression line fell within the 95% CI for AO/PI-measured viability. The coefficient of regression for TB-measured viability was 0.9584, with th e predicted-viability regression line almost entirely outside the 95% CI. T B overestimated the percentage of viable cells, particularly below the 50-p ercent level. CFU-GM frequency correlated better with cell viability measur ed by AO/PI (r(2) = 0.979) than with that measured by TB (r(2) = 0.930). CONCLUSIONS: The AO/PI viability assay is a rapid, highly linear, functiona lly correlated assay that is superior to conventional viability measurement by TB exclusion.