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127 Iodine, 138 Barium, 208 Lead Contamination

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komal2000

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Posts: 11

Joined: Mon Aug 29, 2016 12:55 pm

Location: University of Helsinki, Finland

Post Wed Mar 11, 2020 11:44 am

127 Iodine, 138 Barium, 208 Lead Contamination

Dear All,

I am running multiple myeloma samples and they are Ficoll separated MNCs. I have an issue with iodine, barium and lead contamination. U can clearly see very high contamination of iodine, Barium followed by lead in the rain plot (take a look at the attached file). What is the negative impact of these contamination on the analysis? . I am thinking some of the iodine is from Ficoll separation.


Sincerely
Komal
Attachments
rain plot 2.pdf
Rian plot
(99.8 KiB) Downloaded 78 times
Rainplot1_n.pdf
Rian plot
(90.76 KiB) Downloaded 66 times
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NikPaschal

Participant

Posts: 1

Joined: Wed Jan 30, 2019 10:43 am

Location: Mass Cytometry-CyTOF Lab, Biomedical Research Foundation Academy of Athens (BRFAA), Athens, Greece.

Post Wed Mar 11, 2020 12:36 pm

Re: 127 Iodine, 138 Barium, 208 Lead Contamination

Hi Komal,

Yes I think Ficoll could be a likely source for iodine. Check some previous posts on this issue here

viewtopic.php?f=7&t=405

I don't think it would be much of a problem for the analysis, however it is certainly not ideal for your system/detector. If you have verified that these contaminants are not from your buffers/system and you have more of this batch of sample to process consider extra washes prior to acquisition and dilute the sample a bit more.

regards,
Nikos 8-)

BRFAA CyTOF lab, Athens, Greece.
-----------------------------------------
Nikolaos Paschalidis, Ph.D.
CyTOF Lab
Biomedical Research Foundation
Academy of Athens (BRFAA), Greece.
www.bioacademy.gr/?lang=gr
npaschal@bioacademy.gr
www.linkedin.com/pub/nikolaos-paschalidis/a5/b5/b9b
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EHaasDFCI

Contributor

Posts: 24

Joined: Thu Jun 15, 2017 5:34 pm

Post Wed Mar 11, 2020 7:13 pm

Re: 127 Iodine, 138 Barium, 208 Lead Contamination

Hi Komal,

TO me it looks like the samples could use another wash and perhaps further dilution, depending on your acquisition speed. What was your event rate for this?

The iodine contamination is likely from the ficoll, but it actually looks to be cell specific and shouldn't be an issue at this level with another wash. Barium is also just something that shows up in these sorts of patient samples from time to time, and based on the rain plot, again, it doesn't seem alarmingly high, just dirty. For the lead, that's more intriguing, since I've only encountered lead during runs when air is introduced to the system, but this looks cell specific, so I defer that contaminant. As long as the contaminants stay relatively low and are cell specific, they should not impact the data very much, but watch out for streaking, as this may effect the actual event count and could cause some abundance and oxidation based spillover.

Eric
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komal2000

Participant

Posts: 11

Joined: Mon Aug 29, 2016 12:55 pm

Location: University of Helsinki, Finland

Post Mon Mar 16, 2020 10:20 am

Re: 127 Iodine, 138 Barium, 208 Lead Contamination

Dear Erik and Nikos,

Thanks for the Suggestions and I will follow. For Erik question regarding the events it was 300000. I have one more question regarding combing multiple runs from the same samples in to a single fcs file. After combing the multiple runs of the same sample in to a single fcs file I tried to analyze the data in flowjo. Somehow it is not showing any events. I don't understand why? please check the images attached.


Sincerely
Komal
Attachments
Screen Shot 2020-03-16 at 12.18.41.png
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jimbomahoney

Master

Posts: 73

Joined: Wed Feb 27, 2019 11:21 am

Post Wed Mar 18, 2020 7:55 am

Re: 127 Iodine, 138 Barium, 208 Lead Contamination

Hi Komal,

It's hard to tell from that screenshot, but were the EQ beads added to the sample before running? (It looks like that's a bead gate in FlowJo).

Did you normalise the files before concatenation?

(In CyTOF software, you should normalise, then concatenate. I'm not sure about the CATALYST/ Finck method as the website is down right now, but I seem to remember that allows normalisation after concatenation).

James
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komal2000

Participant

Posts: 11

Joined: Mon Aug 29, 2016 12:55 pm

Location: University of Helsinki, Finland

Post Wed Mar 18, 2020 11:39 am

Re: 127 Iodine, 138 Barium, 208 Lead Contamination

Hi James,

Yes beads were added to the sample before running and also they are normalized before concatenation (Helios). If I use the antibodies (markers) for gating I can see the percentages but for instance if I use beads, time or other parameters to clean up debris, normalization beads etc I don't see the percentages. Do I need to normalize the files after concatenation?.


Sincerely
Komal
Attachments
Screen Shot 2020-03-18 at 13.32.13.png
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jimbomahoney

Master

Posts: 73

Joined: Wed Feb 27, 2019 11:21 am

Post Wed Mar 18, 2020 12:55 pm

Re: 127 Iodine, 138 Barium, 208 Lead Contamination

Hi Komal,

If you're using CyTOF SW, then you should normalise, then concatenate, so you're doing it right.

What version of FlowJo? Maybe it's a problem there? (I can't advise further on that I'm afraid - try updating FlowJo?).

Of course, if you're gating on cells first, you probably have gated out the beads, but if my recollection of FlowJo is correct, those are all indepdent gates in the screenshot, not child gates. In which case, that is weird.

James
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Mar 18, 2020 4:18 pm

Re: 127 Iodine, 138 Barium, 208 Lead Contamination

Hi Komal,

You've shown us rain plots and FlowJo workspace gating pathway screenshots, but you haven't shown us any bivarate plots.

So, let's back up a few steps: if you take your original Raw files (before concatenation, before normalization), put them into FlowJo, and then look at Ir vs Ce140, do you *see* EQ beads in the bivariate plot?

If so, then repeat for your normalized file(s) and repeat also for your concatenated file (or whichever order you did those in this time).

In other words:
1. Find out if the beads are actually "missing", or if your gate is just "off". Both would result in zero beads in your gate.
2. If the beads are actually missing, then you would find out which step made them disappear. For example, most normalization methods allow you to "remove" the EQ beads in the final step, so if that box accidentally got checked, your files wouldn't contain them. Or, if the normalization somehow got screwed up, your beads would still be there, but would be outside of your gate.


Mike
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EHaasDFCI

Contributor

Posts: 24

Joined: Thu Jun 15, 2017 5:34 pm

Post Wed Mar 18, 2020 5:21 pm

Re: 127 Iodine, 138 Barium, 208 Lead Contamination

Hi Komal,

In addition to Mike's suggestions, for your concatenated file, I would try putting your channel of interest (140Ce Beads) on the y-axis and time on the x-axis. Then, customize the linear time axis and change your maximum value to something along the lines of 20,000,000. See if this allows you to gate now and see the statistics. For whatever reason, when concatenating files, then analyzing in FlowJo, the time scale gets scaled up immensely.

Following up on the previous question, what was the event rate that the samples were acquired? This can contribute to more of a 'dirty' signal if you are running the samples at too high of a rate.

Eric
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komal2000

Participant

Posts: 11

Joined: Mon Aug 29, 2016 12:55 pm

Location: University of Helsinki, Finland

Post Thu Mar 19, 2020 8:58 am

Re: 127 Iodine, 138 Barium, 208 Lead Contamination

Dear all,

Thanks for the suggestions. It was bit strange that when I used only concatenated file and customize the axis the problem was not solved. Bead can be seen clearly in the concatenated file but when I do the clean up procedure it was not showing the percentages and also gating.
I tried different sample ( unconcatenated file) and customized the axis then applied the changes to concatenated files then the problem is solved.

Sincerely
Komal
Attachments
Screen Shot 2020-03-19 at 10.56.04.png
solved
Screen Shot 2020-03-19 at 8.37.59.png
biaxial plot
Screen Shot 2020-03-19 at 8.36.49.png
residual gate
Screen Shot 2020-03-19 at 8.35.32.png
Bead gate

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