[Cytometry] Throwing "logs" on the fire
thomas.delohery at verizon.net
Thu May 19 22:07:24 EDT 2011
Mario, we are not in disagreement.
Most of the data we have is from a FACSCalibur and we are currently optimizing a +10-color panel for the Fortessa where everything is more manageable; especially because I can put the lymphocytes into that parallel universe of negative fluorescence. What's with that? anti-photons? Initially, I was concerned my data would disappear due to anti-photon - photon annihilation, but everything appears to be working.
Today I took a closer look at the data to address your response. I looked at a FACSCalibur experiment for FITC conjugated HLA-DR which has always been the problem. Note, for this experiment we had optimized several variables including titering the antibody down to ~ 10-fold less from our earlier attempts. Titrations were based on HLA-DR levels on peripheral blood B cells and we were concerned about detecting HLA-DR on "small" DCs at decreasing Ab concentrations; i.e., newly arrived DCs from periphery as this was part of why we were in the lung. Also, autofluorescence levels for lymphocytes (unstained and iso-controls) were MFI ~ 2 (median FI) because the negative parallel universe is not available on a Calibur and most lymphocytes were crammed on the baseline. MFI for resident alveolar macrophages for this particular experiment were not too bad, ~200+; 3rd decade, 2 logs above lymphocytes. MFI for resident alveolar macrophages stained with HLA-DR FITC was ~ 80% off-scale, piled in last channel, MFI ~ 10,000. Presumably, MFI would be somewhere ~ 20,000 in the 5th decade, 2 logs above macrophage autofluorescence and 4 logs above lymphocyte autofluorescence, just as you surmised. But they are still off-scale, which was the point I was trying to make. Also, resident alveolar macrophages do appear to uniformly express ~10-fold higher levels of HLA-DR than lymphocyte-sized cells in BAL (most, ~80%, are HLA-DR neg.) - the positives do not extend to the 3rd decade. All values cited are approximations/averages from 12 cyno samples.
So there. Ever hear of a guy name Tompall Glaser? Put another log on the fire. look him up in case they scrub the this YouTube link.
On May 18, 2011, at 11:03 AM, Mario Roederer wrote:
> Tom, you've known me far too long and too well to risk asking me to be nice :). Note -- I changed the subject line, since it had nothing to do with the topic.
> I have to take issue with what you state. Just because the autofluorescence runs along 3-4 decades doesn't mean that when you add fluorescence to the system it will extend the range beyond 4-5 decades.
> Look at it this way: Let's assume a lymphocyte (with autofluorescence at 10 units, or first decade) expresses HLA-DR such that it's fluorescence shows up at 10,000 units (or the 4th decade). That's a bright expression pattern already!
> Now let's take an alveolar macrophage (with autofluorescence at the fourth decade, i.e., 10,000 units), and assume it expresses HLA-DR to the same extent as the lymphocyte (same number of HLA-DR per cell). Where does it show up? Not at the 7th decade -- rather, it's STILL in the 4th decade. The fluorescence is now 10,000 (AF) + 10,000 (DR) = 20,000, which is only 1/3rd of a decade above the start of the 4th decade.
> OK, so now you tell me that the macrophages express much more DR than lymphocytes. Fair enough. Do you think they express 10x as much? If so, they appear in the 5th decade -- still only 4 decades above the unstained lymphocytes. And note -- they appear in the 5th decade not because of the autofluorescence, but because they express that much receptor! The AF would be only 10% of their signal!
> My point is that it's wrong to think that because the autofluorescence ranges over 3-4 decades, that the fluorescence signal will range over a much greater range. The range of fluorescence signal is NOT affected by autofluorescence! (Think it through before you disagree with this).
> If you really have a marker that is expressed so brightly as to appear 5 or more decades above lymphocyte autofluorescence, then perhaps you should use a dimmer color (like FITC) to detect it. No need to waste PE on that reagent. And if you do have such a marker (I'd love to know about it!!), then I'd ask how important is it to have the lymphocyte autofluorescence wasting a decade at the bottom of the scale? Why not just put the positive marker on-scale, and if you have to make the lymphocytes reasonably squashed near the bottom -- you'll still be able to detect dim fluorescences on them anyway.
> Bottom line -- autofluorescence doesn't impact need for more "logs". Only if you need to discriminate 5+ decades of true expression level would you need more logs, and no one has ever shown me an example where that is the case.
> As for side scatter -- we have been putting this on a log scale since the beginning of FACS. (Yes, this is how I was trained by the Herzenbergs, who truly are the beginning of FACS!). We've always felt that log SS is a much better way of viewing PBMC or BAL or any mixture of lymphocytes, monos, and neuts, because it gives nice distributions.
> On May 17, 2011, at 9:38 PM, thomas delohery wrote:
>> okay you guys,
>> In recent months I've been working with bronchoalveolar lavage (BAL) samples and the autofluorescence of the various immunocytes in these samples can extend over 4-decades; i.e., unstained lymphocytes in the first decade and macrophages overlapping the 3rd and 4th decades along with some other interesting populations in between. Once you add fluorescent markers to the mix it is virtually impossible to get all the populations on a 4+ log scale. HLA/DR is particularly problematic, esp. when you are attempting to identify subpopulations of dendritic cells other APCs as well as activated T cells. Running the samples twice after changing PMT voltages, redoing compensation, etc., is something I really want to avoid as it makes the analysis less incredibly difficult, not to mention the analysis. I think I need more logs.
>> Also, how about a 1-log scale for 90' side scatter? that would be very useful.
>> now be nice, TomD
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