How do I establish and validate a panel using a viability dye such as 7-AAD? How do I perform compensation for this panel

Viability assessment is essential for accurate multiparametric flow cytometry analysis. The purposes of this assessment include ensuring sample integrity, precise characterization of antigenically-intact (viable) cells, and quantitation and/or exclusion of dead cells (if viability assessment is incorporated within the assay). CAP accreditation requires the presence of "a policy for determining when the percentage of viable cells in each test specimen should be measured" (please refer to CAP Flow Cytometry Checklist 8/21/17; FLO.30610).

There are several methods to assess cell viability, including those applicable for incorporation into the flow cytometry analysis. One of these is 7-Aminoactinomycin D (7-AAD). 7-AAD is a fluorescent DNA intercalating dye, which means it gets attached or inserted between DNA base pairs. It has a spectral emission that can be separated from PE and FITC.

There are few considerations that should be taken when 7-AAD is incorporated into the flow cytometry assay. The cells should be unfixed at the time of staining. Also one needs to reserve the PerCP or PerCP-Cy5.5 channels for 7-AAD. That means that no other fluorescent antibodies can be used in these channels.

With regard to compensation for 7-AAD, no specific measure needs to be taken as 7-AAD-positive cells (dead cells) will be excluded from the analysis. In this way, the 7-AAD channel is designated a "dump channel". This is mainly true in quantitative assays such as CD34 enumeration. In qualitative assays, such as leukemia/lymphoma immunophenotyping, this could be done for the purpose of quantifying the percentage of dead cells to assess sample integrity as required by CAP (utilizing one combination/tube for viability assessment), or for excluding dead cells in each combination/tube. The disadvantage of the latter is imposing restriction on the antibody panel design due to reserving channel for 7-AAD in each tube.

There are several other Q&A topics that deal with viability that provide additional information on this subject:

"How and in what situations should viability of specimens for flow cytometry be assessed?"

"What causes non-specific antibody binding and how can it be prevented?"

References:

Schmid I, Krall WJ, Uittenbogaart CH, et al. (1992) Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. Cytometry. 13:204-208

Njemini R, Onyema OO, Renmans W, et al. (2014) Shortcomings in the application of multiparameter flow cytometry in lymphocyte subsets enumeration. Scand J Immunol. 79:75-89.