Wow! Thomas, I did not mean to stir up such a hornets nest or impugn the CD63 assay. My original comments were mostly directed at Howard's "basic orange 21" assay. I would agree that the CD63 assay of basophil activation is well supported by a large number of publications. We have used the CD63 basophil assay and I have been impressed by the simplicity of the assay and high quality of the data generated. 1. My major point was that assays measuring drops in cellular mediator contents (tryptase, basic orange 21) are inherently less sensitive than those measuring increases (such as histamine release and CD63 translocation). 2. The major strength of the CD63 assay is in measuring degranulation of basophils, as this uses the gating capability of flow cytometry. 3. Using the CD63 assay on cell lines, such as Amy notes, does not utilize this unique strength of the assay. 4. Since the assay has not yet been validated on human or rodent mast cells, I do think Amy is asking for trouble and not getting much added value relative to a traditional release assay. I agree that the CD63 basophil assay is more sensitive than CD203. A question while we have you on the line. About 10% of donors demonstrate a basophil "non-releaser" phenotype when histamine release is examined. Is a similar group of non-responders seen with the CD63 assay? Calman > ---------- > From: Nebe, Thomas C. > Sent: Monday, November 11, 2002 13:08 > To: Cytometry Mail Server (E-Mail) > Cc: CPRUSSIN@niaid.nih.gov; araber@athersys.com; Nebe-Von-Caron, G > Subject: was "degranulation" now "why flow?" > > Dear Dr. Aber, > Dear Dr. Prussin, > > I want to give a few comments about measuring basophil degranulation by > flow and CD63 as my brother forwarded me your discussion on the Purdue > server. > > We have done the classical histamin release test practically in our lab > for a long time (checked two different suppliers) and developed that flow > test because of the drawbacks of the histamin immunoassays. We have tested > several dyes and many surface molecules for basophils and decided to use > gp55, now called CD63 as this approach came up with the best results. A > french group did it independently in parallel (Sainte-Laudy). We also > carefully validated our assay against the histamin release assay. BD and a > swiss company from de Weck just released a copy of that test. > > Our clinical problems to solve were to discriminate bee and wasp venom > allergy and to check for latex allergy, where we had a lot of sesitized > persons among our 4000 employees, where the specific IgE test did not work > properly. > > The ELISA is less reliable because of variations of spontaneous histamin > release and several allergens were proven to contain suffcient amounts of > histamin that we just tried to measure. Contaminating bacteria in the raw > material are able to convert histidin to histamin. The ELISA requires > batch testing and it takes 2 days as you have to convert histamin to a > derivative that the monoclonal can detect as you cannot immunize with > histamin. The day to day variability is considerable. > > Concerning the referees, there are already enough papers out using that > method. Don`t be afraid. Of course, the calculations are different as you > do not measure the same thing. But you could ratio the spontaneous > percentage of CD63 expression and the stimulated % of CD63 if you like to. > The presented conversion from percentages of release into a drop in mean > channels is hard to follow. > > Using a cell line, the basotest kit might be overdone as it addresses > whole blood from patients and CD63 may be sufficient for the homogeneous > pouplation (CD63 has already been used for mast cell lines). We also > compared CD203 from Dr. Buehring that also identify basophils but the > shift in expression density of this marker upon degranulation is not > sensitive enough. The spontaneous histamin release from dead cells from > day to day might be a concern and might favour an antibody staining. > > So, even appreciating the theoretical considerations from Dr. Prussin, > from a practical point of view, the flow assay makes some sense. It might > not be the last answer to the intriguing challenge of clinical tests for > hypersensitivity. > > Best regards > Thomas Nebe > > Dr. C. Thomas Nebe > Facharzt für Laboratoriumsmedizin und Klinischer Chemiker > Institut für Klinische Chemie > Zentrallabor des > Universitätsklinikum Mannheim > Theodor-Kutzer-Ufer 1-3 > D-68167 Mannheim > Tel. +49 621 383-3485 > FAX +49 621 383-733485 > thomas.nebe@ikc.ma.uni-heidelberg.de > http://www.ma.uni-heidelberg.de/inst/ikc > > >
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