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problem with Fluidigm Pd barcoding

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Diane1

Participant

Posts: 7

Joined: Thu Jun 08, 2017 9:55 pm

Post Tue May 01, 2018 9:12 pm

problem with Fluidigm Pd barcoding

Hi All,
We recently are experiencing problems with the Fluidigm Pd barcode staining of PBMC's. I initially noticed that intensity on the rain plot was not great, and when I debarcoded the data I found that we had very low yield. Normally we recover over 200,000 events and with this new lot of barcodes, I recovered from 20-30,000 events. I compared old barcodes to new barcodes and found that I could recover my usual 200,000 events with the old, but the new was only 32,000. Fluidigm sent us a new lot and we saw the same thing. I am wondering if others are experiencing this?? I've been using the Cytof software for debarcoding.

Thanks,
Diane
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nsigal

Participant

Posts: 8

Joined: Fri Jul 25, 2014 5:26 pm

Post Wed May 02, 2018 6:14 am

Re: problem with Fluidigm Pd barcoding

Hi all,
I would like to add to Diane's post and say that we have seen same problem with two new barcode kits lot#0571801 and lot#0861801. Another one that Diane's has mentioned as problematic is lot#111805.
We looked at Pd signal intensities in flowjo and compared new lots with some old lots. It seems that new lots Pd signal intensities are at least log lower then old lots Pd signal intensities and this, I suspect, is the main reason for the low debarcoded yield recovery with new lots. Each one of six Pd in new questionable lots has median intensity below 50.

This low yield recovery is only happening when Fluidigm debarcoder is used and only if "Minimum signal threshold for barcodes" option is checked. If I remember correctly, this option appeared after last software upgrade and is checked by default. If you uncheck "Minimum signal threshold for barcodes" or simply manually debarcode samples by boolean gating, you should gain back the yield. Debarcoding with premessa debarcoder will solve this problem as well.

I have examples of signal intensities from both new and old lots and can post it here if anyone is interested.
Please let us know if you have experienced similar problems with Fluidigm barcode kits recently,

Best,
Natalia
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fcl54643

Contributor

Posts: 21

Joined: Mon Nov 02, 2015 2:13 pm

Location: Greater Philadelphia Area

Post Wed May 02, 2018 12:36 pm

Re: problem with Fluidigm Pd barcoding

Hi Diane,

We have also noticed a substantial reduction in our barcoding yield over the past month (in some instances, 2-3% yield only). Our Fluidigm field specialist suggested that the fixation buffer (Fix I buffer from the Pd barcoding kit) might be the root of the problem, therefore we switched to a different fixation buffer (BD Cytofix, used at a final concentration of 2% PFA). Switching to this fixation buffer only slightly improved the yield (~9%), so I wonder if the real problem is barcode-related.

Any insights would be welcome. Thank you!

Florence
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brferrell

Participant

Posts: 1

Joined: Fri Jul 24, 2015 1:21 pm

Post Wed May 02, 2018 9:53 pm

Re: problem with Fluidigm Pd barcoding

Dear Diane et al.,
My lab has also just run samples with new kits all from lot 0861801 and noted that the barcoded staining was at least a log lower than all previous samples run this year (on kits purchased last fall). We only use our own fixative/PFA mix, not the one that comes in the kits, so this isn't it. I am also not sure if it is a problem bc with the fluidigm debarcoder and not using a min for barcode intensity (unchecking box), we get 97.5% yield with otherwise default parameters. Some cells do pile up at zero for a particular barcode channel (~5%), but I am not sure that is a problem as long as each individual cell can be evaluated for 3on/3off barcode. Maybe someone else has thoughts on whether low barcode signal could o/w cause a problem (assuming you can assign correct barcode).

Cheers,
Brent

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