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

Abstract

BACKGROUND: A reliable, validated method for rapidly determining HPC viability is essential for clinical cell engineering. STUDY DESIGN AND METHODS: A fluorometric cell viability assay using acridine orange and propidium iodide (AO/PI) was compared to the current standard, trypan blue (TB) exclusion. Viable cells stained with AO/PI fluoresce green under darkfield fluorescence microscopy, while nonviable cells fluoresce orange. Mixtures of fresh and heat‐killed bone marrow were prepared and used as viability standards for evaluation of both assays. The frequency of CFU‐GM was determined for each specimen. RESULTS: Cell viability measured by AO/PI was extremely linear, with measured 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‐viability regression line fell within the 95% CI for AO/PI‐measured viability. The coefficient of regression for TB‐measured viability was 0.9584, with the predicted‐viability regression line almost entirely outside the 95% CI. TB overestimated the percentage of viable cells, particularly below the 50‐percent level. CFU‐GM frequency correlated better with cell viability measured 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, functionally correlated assay that is superior to conventional viability measurement by TB exclusion.