This is a good topic for discussion. What is the definition of rare? 1/100000 ? 1/1,000,000? Those of us doing CD34 enumeration are routinely asked to give results sometimes as low as .1%. In fact normal adults have a measurable percentage as low as .02%. When I first started CD34 enumeration 5 years ago I was told you had to have at least 50 of these positive events to be significant. Needless to say we acquire alot of data to get these numbers. 20 years ago I believed the error by flow to be around 2-5%. If you run the same tube 10 times you will get some variance in the percentage you are looking for, particularly in the levels of .1%. The problem occurring is the significance of an increase from a measured .2% to a .4% level. Is this increase real or is it a factor of the instrument. When this number is used to calculate Absolute numbers in a leukopheresis product then it can be significant. Now I have been requested to give numbers lower than .01%. How to make this accurate? Good question. I assume that most populations have some inherent properties to them. They scatter light in discrete patterns and have some sort of phenotype unique to them. The more parameters I use the better the confidence level. If I set all analysis markers by isotype controls then that could make my decision easier, but these give erroneous results if you look at the data carefully. If you collect 500,000 cells and then see a population of 16 are these real???? In practice the low %'s <1% are at best sometimes subjective to the operator's experience. A good question is not only the ability by flow to give these low %'s but how reproducible is it. Some labs are running their samples in doublets or triplets then averaging. This will drive the cost up a bit. Are there any statisticians out there who can answer some of these questions? Jim Houston -----Original Message----- From: Lynn Dustin [mailto:dustinl@mail.rockefeller.edu] Sent: Friday, January 26, 2001 12:39 PM To: cyto-inbox Subject: very rare events: how low can you go? Hello all, 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? Thanks in advance for your help! ********************************************** Lynn B. Dustin, Ph.D. Center for the Study of Hepatitis C Rockefeller University Box 64 1230 York Ave. New York, New York 10021 Phone: 212-327-7067 email: dustinl@mail.rockefeller.edu
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