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Low event counts

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ssivajothi

Contributor

Posts: 29

Joined: Mon Aug 01, 2016 2:31 pm

Post Wed Aug 29, 2018 1:45 pm

Low event counts

Hi all,

I am having a peculiar problem on the Helios which I am struggling to solve.

The event rate has suddenly fallen to extremely low levels- it hovers around 20 events/sec !! The flow rate is steady and the nebulizer does not have any clogs (checked under microscope). There is no loss of vacuum on the instrument either. I did find a cracked sample delivery line (silica) which I thought was the issue, but replacing it did not solve the issue either. I have changed every part I can think of, to no avail.

Tuning is okay and the bead test runs fine, but the cells are definitely disappearing somewhere! Has anyone encountered this issue previously? Any ideas how to resolve it?

Thank you in advance,

Santhosh
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Aug 29, 2018 3:31 pm

Re: Low event counts

Hi Santhosh,

A few questions:

1. You say that the event rate has suddenly fallen to extremely low levels. Does that mean you were running a sample at an expected event rate, then suddenly that *same sample* dropped to 20 events/sec?
2. How are you judging how many event you expect? TC20 cell counter?
3. By "bead test", do you mean running undiluted EQ beads (expect roughly 100 events/sec)?
4. What is your pressure currently? What is your pressure while running normally?
5. What does your background look like (especially in TOF view, not Masses view)? Clean, or streaky?


Mike
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sarahkj

Participant

Posts: 4

Joined: Wed May 04, 2016 2:49 pm

Post Wed Aug 29, 2018 4:22 pm

Re: Low event counts

Hi Santhosh,

We are currently having the exact same problem with the Helios. I had no issues with QC and did not see it until i began running samples. Normally a sample achieves threshold rate within 2 min after starting acquisition and remains relatively steady throughout the run. Last time it took a very long time to reach threshold (if it did?) and then would fluctuate throughout the run much more dramatically than usual dropping from 300 down to 30 without a change in pressure and slowly climbing back up. Sometimes the pressure would even decrease. This was not sample specific. I flushed the lines and did extensive washing with no change. Based on "troubleshooting" i suspect it could mean the nebulizer needs to be replaced but have not tested this yet. Would appreciate any input the forum has on this.

Sarah
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ssivajothi

Contributor

Posts: 29

Joined: Mon Aug 01, 2016 2:31 pm

Post Wed Aug 29, 2018 4:44 pm

Re: Low event counts

@Mike: inresponse to your questions:
1. The first time it happened, the sample was running a ~250 events/s initially then slowly fell off to anywhere between 15-40. Performed some quick checks, but then had issues with plasma ignition itself and then subsequently with tuning failing at Mass calibration. I then changed out all parts with new clean ones, changed O-rings, checked front end alignment etc. Then start-up and tuning went fine. However, from that point forward the event rate has remained low.

2. I estimate event rate by the cell concentration in the sample after counting. I usually run pretty dilute as our samples are mainly from tissue and I aim for 200 event/s.

3. Yes, bead test is running of EQ beads for calibration.

4. Which pressure do you mean exactly? If it is inlet pressure at instrument, it is set to 52 psi.

5. Background looks clean- I see distinct signals when cellular events appear, problem is they are very sparse now due to low event rate.


@Sarah: It is a puzzling issue for sure, but I have a feeling the cause is going to be something really stupid! I just found a brand new nebulizer in the lab so I will try running with that to see if it solves the problem.

All help appreciated!

Santhosh
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mleipold

Guru

Posts: 2086

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Aug 29, 2018 4:58 pm

Re: Low event counts

HI Santhosh,

By pressure, I mean the actual pressure in your system. You can find it in "Status Panel", then toggle "Sample Introduction". You can now see the Status (running, idle, etc), Pressure, and Flow Rate.

An increase in Pressure is usually the first sign a clog is forming. Alternatively, an abnormally *low* pressure can also be a sign of a clog right around or in the PSI flow sensor: a constriction there (particularly just before it) can act like a canyon in a river, increasing the apparent flow rate because it's narrrower. The PSI only applies enough pressure to maintain a 30uL/min default flow rate, so if it's effectively reading the flow rate incorrectly, then it will be too low a pressure.


Did you check backflushing the grounding nut?


Mike
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ssivajothi

Contributor

Posts: 29

Joined: Mon Aug 01, 2016 2:31 pm

Post Wed Aug 29, 2018 6:48 pm

Re: Low event counts

Hi Mike,

yes I have backflushed the grounding nut and all the sample lines. I will repeat the flushes and try the next run with a new nebulizer and note the pressure reading.

Thanks,
Santhosh

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