From: Wayne Shreffler (wayne.shreffler@mssm.edu)
Date: Thu Oct 02 2003 - 16:14:09 EST
I will echo some of Calman’s comments on this. We have some experience looking at basophil activation in the context of food allergic patients who have, like asthmatics, been reported to have higher basophil “twitchiness”. Our strategy is to identify basophils with the combination of CD203c and CD123. CD203c is a great marker, usually quite distinct – especially in atopic individuals, but we’re more confident gating on them with the combination of CD123 and CD203c. We look at CD63 and CD69, as well as CD203c MFI as indicators of activation. Other markers have also been looked at. The minus one controls that Calman suggested are essential for making comparisons. We also stimulate the cells without purification and measure responses pre and post stim. For this purpose, the delta CD63 is much greater, and can resolve two distinct populations of cells, those that have degranulated and those that have not, which can be confirmed by sorting. This is consistent with Knol’s first report of using this as a marker of degranulation. The delta CD203c is never as great and in patients with high baseline levels can be completely absent. Below I've attached an example of CD203c staining and the response to stimulation in vitro. Top w/ stim, bottom w/o. If you really want to assess degranulation, I think you have to use CD63. Wayne On Thursday, October 2, 2003, at 10:46 AM, Prussin, Calman (NIH/NIAID) wrote: > David, > > 1. CD203c has been touted as a "one color basophil degranulation > assay", > because unactivated basos stain at a low level with it and when > activated at > a higher level. Given the low frequency of basophils in peripheral > blood I > find it more aesthetically and scientifically pleasing to use a > multicolor > gating system (such as the one noted in the BD paragraph but could > also use > a CD2, -14, -16, 19 lineage panel plus CD123). Call me old fashioned. > > 2. I have not used CD203c in practice, but my reading of the data is > that > CD63 stains with far greater fluorescence intensity. Comments from the > group > on this would be welcomed. > > 3. From your description, you are attempting to measure the level of > in vivo > basophil degranulation that occurs presumably due to either allergen > exposure or increased basophil "twitchiness". My own experience with > this is > that it is a tough nut to crack. However, a priori, one would want to > choose > the most sensitive assay and the assay that has a larger experimental > window > (measured experimental difference). Given #2 above, if CD63 is indeed > more > sensitive, you would likely have better results with it. However, > there may > be particularities in the biology of these molecules that I am not > accounting for. > > 4. Conjugate choice. Go with PE or APC. May want to consider CD63 > biotin/ > SA-PE if you want to really get signal enhancement. > > 5. Since you are doing these on fresh cells on different days, you > will run > into the problem of how to compare data acquired on different days with > different instrument settings. One option would be to run an isotype > control > for CD63 (other markers the same) and then subtract the isotype control > histogram from the CD63 staining histogram. The beauty of this > approach is > that it is user independent and does not require you to make subjective > decisions about where to place statistical markers. FlowJo has this > subtraction capacity. > > 6. In the Coulter description below, you mention lysing and then > staining. I > would think you would want to stain first and then lyse. > >> ---------- >> From: David Haines >> Sent: Wednesday, October 1, 2003 10:02 >> To: Cytometry Mailing List >> Subject: BASOPHIL DEGRANULATION/ASTHMA >> >> Folks: >> >> Can someone give us advice on basophil >> degranulation? We are proposing to use this >> parameter as an indicator of disease severity in >> asthmatics. An important qualifier: This is for >> research purposes only and will not be used for >> clinical treatment or diagnosis. Our evaluations >> will involve ONLY fresh blood, we will not be >> conducting cell culture of any kind. >> We hypothesize that flow cytometric measurement >> of basophil degranulation levels in whole blood may be >> used to distinguish asthmatics from healthy >> individuals; and may also be used to assess severity >> of disease and effectiveness of (some) therapies. >> >> For each sample of whole blood we would like to >> obtain a number approximating the level of basophil >> degranulation. We are here asking for advice as to >> the most appropriate approach. We are considering >> two candidate strategies: >> >> 1. Whole blood from subjects is lysed in Beckman >> Coulter's reagent native/Kind lyse buffer "Versalyse", >> then stained with CD203c (basophil specific). >> Basophils are gated with CD203c+ . Theoretically, >> since CD203 intensity increases proportional to >> degranulation, histogram overlays of these results >> from asthmatics on results from non-asthmatics will >> demonstrate consistently larger populations of >> brightly-staining cells in asthmatic subjects. No >> idea whether cells undergoing elevated degranulation >> in blood of asthmatics may be distinguished as >> discrete clusters, or form or "schmeers". >> >> ADVICE NEEDED: Has anyone distinguished populations >> of weakly CD203c+ cells (i.e. low degranulation level) >> from strongly CD203c+ cells (i.e. high degranulation >> level)? >> >> Alternative approach (contributed by Holden Maecker of >> BD): >> >> 2. A new application from BD using CD63 expression >> to measure basophil degranulation in response to "test >> stimuli" is offered. Surface expression of this >> antigen is upregulated in certain classes of cells >> upon activation of neutrophils, monocytes, >> macrophages, endothelium and activated basophils. A >> cocktail of anti-CD63 FITC, -CD123 PE and -HLADR PerCP >> may be used to quantitate levels of degranulating >> basophils following stimtheir stimulation in culture. >> >> ADVICE NEEDED: In absence of external stimulation >> (which we are not going to do), how well will this >> approach distinguish subpopulations of basophils based >> on their level of degranulation? Does anyone have >> any experience with this? >> >> NOVEL APPLICATION: Could CD203c be used to gate on >> the basophil population - and CD63 used to identify >> subpopulations of basophils at various levels of >> degranulation? This question is directed >> specifically at Holden Maecker (BD) and Hector Pulido >> (Beckman Coulter). Thanks! >> >> Any responses will be greatly appreciated. >> >> Many Thanks >> >> David D. Haines & >> Fadia F. Mahmoud, Ph.D. >> Department of Medical Laboratory Technology >> Kuwait University Faculty of Health Sciences and >> Nursing >> 31470 Suliebikhat, Code 90805 >> State of Kuwait >> ddhaines2002@yahoo.com >> fadia101@yahoo.com >> >> >> >> __________________________________ >> Do you Yahoo!? >> Yahoo! SiteBuilder - Free, easy-to-use web site design software >> http://sitebuilder.yahoo.com >> >> >> Wayne G. Shreffler, MD, PhD Assistant Professor Department of Pediatrics Division of Allergy & Immunology Mount Sinai Medical Center, box 1198 One Gustave L Levy Place New York, NY 10029 212-241-7244 voice 212-426-1902 fax
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