How many cells do you need to reliably detect a population of interest?

The answer depends on the confidence you need, and the variability you can accept. Variability is expressed by the coefficient of variation (CV) is simply the standard deviation divided by the mean. The higher this number, the more “variable” the measurement. The lower the number, the less “variable” the measurement. Intuitively, a population that appears a lot, say 10% of the time, needs fewer cells than a population that occurs 0.001% of the time.

Luckily, this question has been addressed in the work of Keeney et al. To wit, “for cell-based assays such as flow cytometry, a simple calculation can be used to determine the size of the database/sample that will provide a given precision: r = (100/CV)2; where r is the number of events meeting the required criterion, and CV is the coefficient of variation of a known positive control.”

For a population that’s at the 0.1% level, you need 10,000,000 events to detect with a 1% CV, and 400,000 events to detect with a 5% CV. And using Keeney’s formula, you need 10,000 events of that specific population at the 1% level, and 400 events at the 5% level.

Intermediate Monocytes (inMono) cells are 0.47% of non-granulocytes in Teiko’s internal data. We would need 188 inMono cells to get past a 5% CV, and we actually collected 302 cells in our internal data. As a result, we ended up with a 4.63% intra-run CV. In case you are wondering, all populations measured using Teiko’s standard panel have met the accepted CV criteria of 25-30%.

For more details and to check out the full calculations, read our article.