What are the reason for higher intra-assay variability in measurement of neutrophil oxidative burst using zymozan or PMA induced chemiluminescence?
All assays will vary in terms of output. You simply cannot expect same results from all but different assays.
Dear Nader there are two major mistakes that are encountered. One is to believe that the sample used are homogeneous amount of neutrophils, same age same response, which is not the case. Old neutrophils respond less than young neutrophils. Further the assay can vary depending if the blood donor is smoker, it is older or younger, it is taking antibiotics, or even anti oxidants. So it is not simple to have a standard value, you allways have a range which is more suitable to interpret mean channel fluorescence intensity in which case tolarable differeneces are amoung 10 %
regards,
Nader, I agree with Juan about donor variability. There are other sources of problems too. PMN are very fragile cells that are easily triggered to undergo degranulation and resp burst. As a result, you must be very careful to avoid this during PMN purification and storage before use in your assay. All reagents and plasticware must be endotoxin free, very fresh blood must be used, cells must be used within an hour or two of isolation, cells must be stored on ice in calcium magnesium free buffer with EDTA before use, exposure to free hemoglobin or to platelets must be minimized or prevented. For these reasons, it is much easier to use cultured cells rather than isolated PMN.
Also, the source of plasma for opsonizing zymosan should be kept constant from experiment to experiment. We used purified plasma from a single donor that was aliquoted into small tubes, stored at -80c, thawed and used once only and not re-frozen. PMA must be made up fresh each day.
Regards
Thank you Robert for the tips. I am using whole blood not isolated neutrophils at this time, but I can see that even in whole blood sample how different handling may cause different levels of oxidative burst.
Never heard of anyone using whole blood for oxidative burst studies. No wonder you're having so much difficulty getting consistent results with your assays. When you add PMA or zymosan not only are PMN being activated but so are platelets, eosinophils, basophils and monocytes. And when each cell type is triggered it's releasing further additional factors that will cause additional triggering of all cell types that are present in the mixture. As a result, you don't really know what is the precise trigger that's causing oxidative burst nor will you know which cell types are responding. This experimental system is easy to set up but extremely difficult to control and interpret.
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