R. Thomas Leonard wrote: >Question from a novice: > >There are several papers which describe ways in which cell concentration >can be determined using FCM. When quantifying cells, one must identify >"events" which are assumed to be the cells of interest. How does one >validate, quantitatively, that the events are in fact the cells of >interest. For example, if we count 1x10E6 bacteria, how do we know that >there aren't 9x10E5 bacteria and 1x10E5 small fungi that scatter and >fluoresce in a similar fashion? > >My assumption is that one would need to program the cytometer to sort X >events believed to be cells of interest. We would then need to quantify the >cells using fluorescence microscopy. Any suggestions? Why hold the flow cytometer to a higher standard than the impedance (Coulter) counter? The latter will count as cells anything with a signal set above threshold; if you try to do a "white blood cell count" by putting in diluted whole blood treated with an agent that nominally lyses red cells but not white cells, you will get a falsely high count if some of the red cells are not lysed and a falsely low count if all of the red cells and some of the white cells are lysed. You may have better luck discriminating red from white cells by eye using a hemocytometer, but you are then faced with imprecision due to dilution errors and counting statistics. And, if you had a lot of giant platelets in the blood, you might count those as white cells. So, the first principle of cell counting is to have a way to tell the cells you want to count from everything else. Or almost everything else; even if you count 10,000 cells, your precision is limited to 1% by counting statistics, which means it doesn't matter that much if you count ten things that aren't the cells you want in with the other 9,990 cells you do want. While sorting is a good way to confirm that you are counting what you think you are counting, it isn't absolutely necessary. If you see clusters in flow data that represent roughly the same percentages of a mixed population as you can count by eye, you can generally assume that each cluster represents one of the visually distinct elements of the population. With both eukaryotic and bacterial cells, you do have the problem that an "event" may represent either one cell or an aggregate of two or more. If you plate bacteria on agar, to count "colony forming units", you have the same problem; unless you scan the whole plate at high magnification to start with (and nobody does), you're not sure whether a colony arose from one organism or several stuck together. From a philosophical point of view, you can only be sure that your cluster of bacteria is not contaminated by "stealth fungi" if you sort everything every time. Nobody does that, either. What you do need to worry about when trying to do absolute counts with most fluorescence flow cytometers is figuring out the volume of sample you have analyzed in the process of counting however many cells you count; most of these instruments do not deliver the sample with a volumetric pump. The commonest way to calculate volume is to add beads at a known concentration to the sample; you first need to count the beads with an impedance counter or hemocytometer, etc. etc. - or pay for known numbers pre-aliquotted by the manufacturer. But it does work. -Howard
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