You could increase the time delay to acquire the sample, which would give more time for the machine to acquire the right number of beads. Make sure you vortex the beads profusely before pooling the singleplex and after pooling.

Using a magnetic kit provides better intra-assay reproducibility, is easier to perform and you have no problem with a "bad" well because the filter integrity was affected by the vaccuum.

Make sure the beads fall into the right gate or region and that the doublet discriminator (DD) gate is set to the correct value for the kit.

Hope it helps,

Michel.

Thank you for your answer. I will try your suggestions.

I have had this type of problem and each time it has to do with the probe. Not the height, but it may get a partial clog. I have had to stop the run, take out the probe and sonicate or flush it wih a 1cc syringe and needle, put it back on and adjust the probe height. You then have to restart the run. You will loose one result but it runs well again and there are no low bead counts. This happens mostly if I am running anything sticky like blood or plasma in my samples. The other suggestion is to add a wash in one or two of the welsl on the plate during the run so that the probe gets washed out occasionaly with sheath fluid to help keep it clean., I hope this helps.

Thanks a bunch Linda. I understand what you mean about the Probe clogging and the subsequent sonication / flush clearing it up. What is happening though, with my assays is that I get a read out for each well but not necessarily for each of the 11 analytes I am looking at. For example, some wells in one assay plate may not show read out for IL4, IL5 and IL13 but show the remaining. In a nother assay plate on another day, IL-4, IL-5 and IL-13 may show up along with some others but IFN-g or IL-10 may not show up. Also there is no consistency in which analytes are detected and which are not. In a particular well, have you found that some analytes are detected and some are not, in regards to bead counts? 

I have found that some analytes such as Il-6 will collect more slowely then others but I have not experienced them not showing up.  Set the instrument to collect for the longest time period and check all the kit settings to make sure they are correct when setting up the instrument.  I imagine it is very frustrating since the kits are expensive and your time in valuable.  One final question are you using premixed bead kits are purchasing them individually?  If they are premixed kits this definately should not be happening since there would be no user error when making up the bead solutions.

I am using single plex kits and pooling for each assay. There is that inherent variable in doing this step and I have standardized the steps down to seconds, use the exact same pipettes each time.

If you look at this screenshot attached file, you will see what I mean. Some regions do not show any beads. 

I will try playing around with the time as you suggest and double check the instrument settings and kit settings are all correct. 

Kind regards

You could try using the beads of the problematic cytokine in a singleplex assay to see if it's a problem with the beads or the mixing/recognition of the beads.  If you still have this problem when you run as a singlplex then II would contact the manufacturer and ask about this lot of beads.  I had a premixed 8-plex assay in which IL-8 did not show any beads.  It was a problem with the lot of the kit and was sent a free kit as a replacement.  Good luck,

Thanks Michel, I am setting up a single plex with the problem analytes. You are correct if the single plex also are a problem then it will definitely be the kit, otherwise handling error :-(