Luminex microarray cross-talk problem

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Luminex microarray cross-talk problem

Postby Carbon Copy » Nov 03 2006 8:24 pm

Does anyone have hands-on experience in Luminex liquid microarray? Working on development of Luminex-based assay I have faced a problem of microspheres cross-talk. By another words there is a misreading of certain microspheres regions. I use Bio-Rad Bio-Plex machine and Luminex FlexMAP beads. Could it be a hardware or rather analyte problem? Any ideas?
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Postby 2fluffy » Nov 06 2006 10:08 am

Hi. I'm also developing a Luminex assay. What exactly do you mean by "microsphere cross talk". What value are you using to get your data? You should always use the median to remove any outlier carryover from well to well.
If you could explain in more detail i'll see if I can be of any help - otherwise i would get in touch with Bio-rad technical support.
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Postby Carbon Copy » Nov 06 2006 2:30 pm

By cross-talk I mean a false-positive signal for region different than authentic microspheres region. For instance, beads #73 have been read as #22, or beads #44 have been read as #32. It happens than a single analyte (beads type) is present in a reaction analyzed, but machine is set for analysis of two or more analytes. Sometime such false-positive reading can be really intense and even higher than reading for authentic region. By intense I mean thousands of MFIs. It happens on some kind of random manner, not in all wells, that is why I am starting to think that it could be an optical system misalignment problem rather than analyte problem. If it were beads, they would be misread always in all wells. I have never thought that carryover from well to well could be a possibility.
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Postby 2fluffy » Nov 07 2006 9:58 am

Do you keep the beads out of the light? Could they be being photobleached? I would definately get in touch with the suppliers to discuss this. Bead carry-over is only ~0.5% so the median soon factors this out.

Good to know there is another Luminex user on the forums - I'm sure I'll be asking your advice soon!
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Postby Teuchter » Dec 04 2006 11:52 am

I struggle to see that this is cross-talk - bead 73 and 22 are at opposite ends of the CL1/CL2 scatter. I frequently see beads being read in the 'next-up-right' region - for example bead 22 being read as bead 30, but can eliminate this by gating appropriately.

What DD gate dimensions are you using?

A small point - why are you measuring something that isn't there (by that I mean why have the machine set to look for more than one region, if all you have is one region)? If you only select the bead region(s) that you have in your assay, the fact that some beads might hit elsewhere is irrelevant.

In terms of carry over from well to well - this is indeed a common phenomenon, but, as 2fluffy mentions, reporting the median eliminates it almost completely. We have tested the extent to which well-to-well carry over can occur - we have seen beads carried over in the next 4 or 5 wells, although it is usually only 2 or 3. On one occasion, we saw one bead counted 16 wells after the well containing that bead region.

Is the Luminex passing calibration and control? If yes, you don't need to worry about optical alignment. We have had machines transported all over in the back of cars, etc, and have yet to see the lasers fall out of alignment.

There are several reasons why 'beads' appear elsewhere - some of them are:
Air in the system - see protocols for correcting this
Particles in the buffers - filter everything
Damaged/bleached beads (rare) - can be checked by running surplus beads through the Luminex
Sample/Matrix effects (rare)
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Postby Carbon Copy » Dec 06 2006 5:28 pm

Thank you, guys for replies. As it appeared, 2fluffy was right in his/her assumption that it was a carryover problem. It was indeed a carryover from well to well. I just was confused by the origin of false responses, which I obtained. But, with your help I have finally figured out what was wrong. I was analyzing different samples with different analytes (beads regions) in the same run. In order to do so, I set the machine for all those regions. Whereas each sample included only single analyte. By another words, they were uniplex, but not multiplex reactions. During analysis I was getting readings from other regions, different from beads in the reaction. Mistakenly I thought it was a cross-talk problem. But, in fact it was carryover or contamination. By default set Bio-Rad Bio-Plex software does not show a number of beads analyzed in a well. Only when I opened additional tabs on the panel in Raw Data Report I could see that false responses came from low number of beads carried over from previous reactions. The only thing that still confuses me is the magnitude of fluorescence response from those contaminating microspheres. Even when number of carried over beads was low - tens or singles, the MFI was as high as from hundreds or thousands. I believe, the software takes an approximation and interpolate the fluorescence from that amount of beads to fluorescence from 100 beads (as reading setting). I think it’s wrong and needs to be addressed by Bio-Rad. Machine should not show fluorescence if the number of beads is less than set by user (100 in most cases). Instead it gives an error messages 1 and 4 (low beads number and reading problem) and interpolates fluorescence readings. Anyway, I was happy to solve that puzzle. Thanks!
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XMaP technology

Postby elix71 » Jul 18 2008 9:15 am

Hello,
I am developing protocols of immunoassays with my antigens on Luninex technology in single Plex and I would like ask some technical suggestion about beads aggregates forming during the incubation:
I know that is always recommended to filter sera before start the incubation but sometime the sera amount is so small and it's colud be difficult !
Well, Do you Know if centrifuge the sera is recommended? I noticed a big good difference with this procedure but actually I don’t know if could be some differences about the amount of total IgG express in FI.
Thanks for your cooperation.
:D
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Re: Luminex microarray cross-talk problem

Postby fcs » Jul 23 2009 1:30 am

Carry-over on the Luminex platform should be negligible given a good bead count (> 50). Here is a nice post regarding carryover or the bleeding of beads from one well to the next with the Luminex system.

I am a technical suppport member of MiraiBio so I would be more than happy to address any issues/problems regarding the Luminex hardware or platform.
Last edited by fcs on Jul 23 2009 4:06 pm, edited 1 time in total.
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Re: Luminex microarray cross-talk problem

Postby r.rosati » Jul 23 2009 3:56 pm

Fcs, please note that by our policies if you are a member of Miraibio you must state so - if you are, and fail to declare it, your post will be flagged as spam and your IP might be banned.
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Re: Luminex microarray cross-talk problem

Postby mdfenko » Jul 23 2009 8:22 pm

fcs wrote:Carry-over on the Luminex platform should be negligible given a good bead count (> 50). Here is a nice post regarding carryover or the bleeding of beads from one well to the next with the Luminex system.

I am a technical suppport member of MiraiBio so I would be more than happy to address any issues/problems regarding the Luminex hardware or platform.

i approved this post because it points the way to resolving these problems, but, this topic is 3 years old. i don't expect that the original poster is going to be helped by this response.

try to be current.
talent does what it can
genius does what it must
i do what i get paid to do
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Re: Luminex microarray cross-talk problem

Postby fcs » Jul 23 2009 8:50 pm

mdfenko wrote:
fcs wrote:Carry-over on the Luminex platform should be negligible given a good bead count (> 50). Here is a nice post regarding carryover or the bleeding of beads from one well to the next with the Luminex system.

I am a technical suppport member of MiraiBio so I would be more than happy to address any issues/problems regarding the Luminex hardware or platform.

i approved this post because it points the way to resolving these problems, but, this topic is 3 years old. i don't expect that the original poster is going to be helped by this response.

try to be current.


Hi,

I posted it because it is a legitimate problem that other Luminex users are facing and not just the person who posted it. This information can be very useful for people who are trying to find an answer to the issue through search engines. While the original post is old, the problem is still current and will be as long as the Luminex technology is still in use. As of today, the technology is healthy and growing.
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Re: Luminex microarray cross-talk problem

Postby r.rosati » Jul 24 2009 6:18 pm

Fcs, of course every user's knowledge is precious and you're more than welcome.
I'm just pointing out, as I wrote here and about your other post, that if you're a member of Miraibio and do not declare so when commenting on their products/website, then you're risking a ban. If you're just enthusiastic over this company (as much as to spend your only two posts here advertising their products and website), but you're not working for them, then things are perfectly ok.
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