Julie Pribyl writes- Does anyone have insight into the problem below from a summer student using flow cytometry? Our lab is currently researching how high cholesterol levels affect the diffusion of oxygen through red blood cells. My job for the summer will be trying to find a way to calculate an average surface area, average volume, and topology of the red blood cells. Will FSC and SSC data provide enough information? It is essentially impossible to get the information from FSC and SSC. Flow cytometric measurements of light scattering at two angles are used to derive fairly accurate values for red cell volume in the clinical hetamology counters made by Bayer (formerly Technicon). However, the red cells must be subjected to a chemical treatment which transforms them from their native biconcave disc shape to spheres without changing volume (the process is called isovolumetric sphering) in order to prevent the normal asymmetry of the cells from producing spurious scatter measurement values. Thus, while the volume can be measured, the topology is lost. Even then, the volume measurement is only possible because rigorous standardization and calibration procedures have been developed, and we don't have anything like that for FSC and SSC (although some work has been done on FSC and bacterial volumes). It's easy to collect a lot of FSC and SSC values from cells under different conditions, and hard to derive reliable quantitative information from them, which doesn't necessarily stop people from publishing. This is a really, really hard problem. Solving it is well beyond what could or should be expected from a summer student; it could occupy several Ph.D. students for a number of years. I would think the grosser changes might best be picked up by a combination of volume measurements using a hematology counter and scanning EM combined with image analysis. -Howard
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