Hi all I've been asked by one of my workmates about the use of screening patient sera for anti neutrophil cytoplasmic antibodies (ANCA), by flow. The idea is to screen out all the negatives and retest the positives either by ELISA or IF on neutrophill slides. I know that this is possible but I have a few queries; a) What is the best bethod to obtain and store neutrophils for screening (there needs to be minimal activation of the neutrophils!). b) What is the best method for permeabilising the cells, in house or commercial products? and lastly c) Best detection method: Fab2 FITC anti IGG or would it be better to use a FAB2 biotin, followed by streptavidin FITC, I would appreciate any ideas on these or any other aspects of this test. Very many Thanks John Culkin Dept of Immunology Royal Liverpool Hospital
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