Lynn Dustin wrote- "This question arises from a rather heated discussion following a seminar yesterday, in which the speaker claimed that flow cytometry is not useful for analyzing cells that are less than 1-2% of the starting population. I am sure that with all of the sorting, multicolor analysis, and multiparameter gating that people do, we can prove this assertion wrong. If you have experience or publications with analysis of events well below 1% of the starting cell population, could you please share some examples or references?" The assertion is wrong. With care, one can detect cells at frequencies of 1 in 1 to 10 million; this involves staining the cells of interest for multiple markers and staining the cells not of interest with a cocktail of specific antibodies all labeled with the same color (one not used for the cells of interest). Additional software and hardware tricks are used to minimize sample carryover and neglect events which appear to involve clumps of junk being dislodged from the instrument's tubing. For references see: Gross H-J, Verwer B, Houck D, Recktenwald D: Detection of rare cells at a frequency of one per million by flow cytometry. Cytometry 14:519-26, 1993 Gross H-J, Verwer B, Houck D, Hoffman RA, Recktenwald D: Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 10**-7. Proc Natl Acad Sci USA 92:537-541, 1995 Detection of residual disease in leukemia by multiparameter flow cytometry is generally accepted to be accurate to one cell in 10,000 nucleated cells, and this is done routinely in a number of clinical laboratories. All of that said, it is true that if you want to get a good statistical picture of your cells of interest, i.e., if you'd like an FCS file of 1,000 of those cells present at one in one to ten million cells, you'd have to analyze one to ten billion cells to get the data. On a high-speed (100,000 cells/sec), that would take hours; on a slower instrument, you'd have to run for a day or more. So a core facility manager (and your fellow users) might want to dissuade you from getting into that line of work. -Howard
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