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

Transfusion. 2000 Jun;40(6):693-6. doi: 10.1046/j.1537-2995.2000.40060693.x.


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.

Publication types

  • Comparative Study

MeSH terms

  • Acridine Orange / analysis*
  • Acridine Orange / metabolism
  • Bone Marrow Cells / cytology
  • Bone Marrow Cells / metabolism
  • Cell Membrane Permeability
  • Cell Survival*
  • Evaluation Studies as Topic
  • Fluorescent Dyes / analysis*
  • Fluorescent Dyes / metabolism
  • Hematopoietic Stem Cells / cytology*
  • Hematopoietic Stem Cells / metabolism
  • Humans
  • Microscopy, Fluorescence
  • Propidium / analysis*
  • Propidium / metabolism
  • Sensitivity and Specificity
  • Staining and Labeling / methods*
  • Trypan Blue / analysis*
  • Trypan Blue / metabolism


  • Fluorescent Dyes
  • Propidium
  • Acridine Orange
  • Trypan Blue