We have just run a Luminex 9plex assay (incl.IL-1b, -2, -4, -5, -6, 8, -10, IFN-g, TNF-a) for a clinician group. Standards are perfect but samples are poor. The person who got the serum said they centrifuged the blood immediately after collection (at 1000g for 5min), so I think of insufficient clotting. Do you think this might be a reason for poor results? Part of the samples have been stored at -86°C for more than 6 months and part were only 3 weeks old. Literature would be appreciated if available.
Hi Senay,
What do you mean by "samples are poor". Do you have a good bead count? are the beads distributed in the right bead regions? a high CV? low concentrations?
What is your assay diluent and is it the same between standards and samples?
Also, were the samples fixed with PFA?
With these answers it will be easier to help you.
Cheers!
the bead counts were quite low for all samples, so all of them were outside the std range and cannot be calculated. however the signals are collected in correct regions. assay diluent was the same for std and samples. (R&D Systems RD6-40 for serum samples). the samples were fixed with Sigma's cell freezing medium-DMSO (code: C6164). thanks in advance for your additional comments...
The cell freezing medium does not fix the sample, it only acts as a cryopreserving media for cells. It contains DMSO which you should avoid when using luminex beads. See the following link: http://www.luminexcorp.com/prod/groups/public/documents/lmnxcorp/incompatible-buffers-solvent.pdf
For luminex experiments, you are interested in the supernatant (serum), not the cells. You pellet the cells and keep the supernatant (serum) for analysis. You don't have to add anything to the serum before freezing it, except if it contains pathogenic agents. From what you are telling me, is it possible that you analyzed the cell fraction? This could explain why you have weird results. If it is the case, I hope that you kept the supernatant fraction.
If I am mistaken and you have analyzed the serum fraction but DMSO was added to it then solvent incompatibility could also explain your weird results.
Hope this helps,
Thanks alot michel, it really helped alot! I was not involved in the sample prep side. The clinician was told by a company rep that he had to add this freezing medium in the samples so that he can preserve the sample longer. Obviously this resulted in the loss of precious samples. At least he can avoid to use this in later studies. Thanks again!
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