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 <http://www.ma.uni-heidelberg.de/inst/ikc>
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:26:29 EST