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Do we all need to use WB/CAS protocol?

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BarbaraFazekas

Participant

Posts: 4

Joined: Tue Jun 18, 2019 7:31 am

Post Thu Jun 20, 2019 5:56 pm

Do we all need to use WB/CAS protocol?

We’ve been chasing the source of the Helios cell signal artifacts that have been “solved” by the use of wide-bore and CAS. Clearly not all machines suffer from these artifacts, and not all machines that do manifest them suffer from them all the time.

The main characteristic of the artefact is that it is frequently time-dependent within a single sample. Unfortunately this is not the way that most data are presented. We also see that the artefact may be masked by the routine 50sec acquisition delay that most users employ.

We read with great interest the recent report from Lee et al in BioRXiv and were intrigued to see the differences in the severity of the artefact in different machines. We had the impression that these artifacts were relatively stable, but analysis of our own data over a period of several years has shown us that they are not in our hands. Importantly they are NOT user or sample specific. They are clearly related to the state of the Helios – we can see the quality of our data declining over the course of months.

So what controls the difference? We first tried adjusting the Z axis, which is not changed during the standard tuning that adjusts X and Y. We can move the Z of the sample injector and the entire torch body. We saw no real differences in the frequency of the artifacts (see attached file).

So what has changed whether the artefact appears or not?
We have only 2 real pieces of data that relate to what caused the artifacts to disappear for periods of up to months. The first is when we had the vacuum hose replaced. The second is when our interface vacuum pump was purged. Both these data points indicate that it may be the vacuum level that is crucial, and that is what we have been chasing recently.

When our machine was installed, the interface vacuum (plasma off) was 4 x 10^-4 (and the sheet said specs were < 7 x 10^-4). At the PMs over the last 2 years, it crept up to 8 x 10^-4 (on a new report sheet that no longer gave a spec value), then 9.9 x 10^-4 in October 2018 and currently about 1.3 x 10^-3. We just had the pump replaced, and the value is now 1.5 x 10^-3 – so there are clearly other elements in the pathway that need to be fixed as well.

What I would like to know is what the vacuum levels are on other machines, with, if possible, correlation with the occurrence of the artefact at any time on a particular machine. I have the impression that most users have now switched to a WB plus CAS and may no longer be seeing the artefact – but if you have not replaced any vacuum components since changing over to WB/CAS, your vacuum values will still be relevant. Probably the most important are the values BEFORE the plasma is turned on in the morning – they are on the LEDs behind the door on the LHS of the machine.

BTW when the artefact was a major problem before our recent vacuum pump purge, we saw the same artefact after a full day’s running with WB plus CAS. This indicates to me that our machine is still performing suboptimally.
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BarbaraFazekas

Participant

Posts: 4

Joined: Tue Jun 18, 2019 7:31 am

Post Fri Jun 21, 2019 4:24 pm

Re: Do we all need to use WB/CAS protocol?

here is the attachment from the previous post
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BarbaraFazekas

Participant

Posts: 4

Joined: Tue Jun 18, 2019 7:31 am

Post Fri Jun 21, 2019 9:34 pm

Re: Do we all need to use WB/CAS protocol?

this is not the attachment because somehow I can't insert it into a message!
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BarbaraFazekas

Participant

Posts: 4

Joined: Tue Jun 18, 2019 7:31 am

Post Sat Jun 22, 2019 9:41 pm

Re: Do we all need to use WB/CAS protocol?

Now the attachments are below the site limit
helios artifacts part2_compressed (1).pdf
(738.03 KiB) Downloaded 131 times

Helios artifacts part1_compressed (1).pdf
(1 MiB) Downloaded 128 times
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AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Wed Jul 17, 2019 8:12 pm

Re: Do we all need to use WB/CAS protocol?

Hi Barbara,

Just a note that we're typically seeing (at least) two distinct artifacts on our instruments.

One is the arifact that we reported in our pre-print (https://www.biorxiv.org/content/10.1101/600130v1), which is reduced signal intensity on a subset of cells in a sample which also manifests as higher population specific-marker CVs. This certainly seems to be instrument and sample dependent, however it appears to not be time dependent as far as we've seen (i.e., the artefact is stable over the course of acquisition. This is the artifact that seems to be largely resolved by switching to the WB+CAS (in the case of instruments where it is present in the first place).

A second set of arifacts is the time dependent loss of signal, and a third is channel-specific increase in signal background. These seems to be separate issues from the first artifact in that they do not seem to be improved by switching to the WB+CAS protcol.

I think your idea of a potential relationship with vacuum levels is interesting, and it's a shame that this is a challenging parameter to perform automated tracking on.

Adeeb
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC
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mleipold

Guru

Posts: 2095

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Thu Jul 18, 2019 9:58 pm

Re: Do we all need to use WB/CAS protocol?

Hi Barbara,

I finally remembered to check our machines' vacuum levels this week, to compare with yours.

Machine 1:
start of day: 1.76e-7, 5.44e-4
4hr later: 4.19e-7, 1.72e-2

Machine 2:
start of day: 5.92e-8, 4.45e-4
6hr later: 3.51e-7, 1.57e-2


Mike

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