My two cents... The use of K-S or other appropriate statistics to the comparison of two histograms (e.g. sample A versus sample B) will reveal the true statistical difference (i.e. either a p-value or a confidence interval). Given the high number of events (typically 5 - 10,000 or more), the uncertainty of the mean (aka. standard error of the mean) is very very small, even though the distribution of each histogram is broad and overlapping. Consequently, even the smallest difference in mean arbitrary fluorescence of sample A versus sample B, is perceived as statistically significant. Therefore, as reported to this list, running the same sample twice, will often lead to statistical significance when comparing results of the two runs by the K-S test. However, this is not what we really want to know !!! What we want to know is: as evaluated by measurement of fluorescence, does sample A belong to population of cells (or individuals) different from that of sample B ? To answer this question we have to know the variation in mean (geometric mean or median) fluorescence when running the same sample multiple times on the flow cytometer. Next, to compare the difference of mean fluorescence of sample A and sample B with this uncertainty of the mean. If the former is bigger than the latter, it may be concluded that sample A and sample B belong to two different populations of cells. Formally, this may be expressed as: Mean fluoresc. sample A - mean fluoresc. sample B > SQRoot 2 x standard error of the mean (of fluorescence measurement) To conclude, the relevant comparison is not that of 2 means from 2 distributions of events (each from a single run), but the comparison of 2 means from 2 distributions of means (from multiple runs). In the latter case, knowledge of a general uncertainty of measurement of mean fluorescence (CV% or standard deviation), may substitute for the repeat measuring of the 2 samples, according to the abovementioned formula. Please note: The above does not take into account the variations introduced by staining, lysing, washing, etc, only the crude variation in fluorescence measurement by the flow cytometer. Please comment, Ulrik Sprogoe-Jakobsen, M.D. Dept. Clinical Immunology Odense University Hospital 5000 Denmark E-mail: ulrik.sprogoe-jakobsen@ouh.dk
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