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Use of Cryopreserved Buffy coat or PBMCs (derived from?)

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Ajaysud

Participant

Posts: 1

Joined: Wed Nov 02, 2016 11:38 am

Post Wed Nov 02, 2016 3:47 pm

Use of Cryopreserved Buffy coat or PBMCs (derived from?)

Has anyone had experience of running a starting cryopreserved buffy coat through the staining protocol and then the Helios? We have tried it and it tended to cause lot of blockages in the Helios device. Has anyone else had this experience, and found a way of overcoming it?? I was wondering also if there was a method/protocol to extract/clean-up buffy coat and extract PMBC's which may be easier to run through the Helios?

Any advice appreciated.

Thanks
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mleipold

Guru

Posts: 1778

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Wed Nov 02, 2016 5:24 pm

Re: Use of Cryopreserved Buffy coat or PBMCs (derived from?)

Hi Ajay,

The HIMC regularly runs cryopreserved PBMCs through the CyTOF; we don't do much work with buffy coats: http://www.conversantbio.com/blog/3-rea ... -isolation

But, we've run samples from whole blood, as well as bone marrow and disaggregated tissue.


The bottom line: the Helios PSI unit clogs like crazy. We've been fighting this battle for almost 18 months. My understanding is that Fluidigm is working on something, but it's been more than two months since I've heard from them on this topic. At CYTO 2016 in Seattle in June, the vast majority of the people (worldwide) with Helios that I talked to have stated that they have the same clogging problems. Some users are avoiding the problem by using the PSI unit only for Tuning, and to run their samples with the third-party SuperSampler.


We've been able to come up with some guidelines to *minimized* clog development:
1. Sample cell concentration. On CyTOFv1 or CyTOFv2, we would typically dilute to 1M/mL (as measured by Biorad TC20 cell counter or similar device that can work in bright-field). Now, we dilute to no more than 0.8M/mL for PBMCs (corresponding to 200-300 events/sec), and no more than 0.5-0.6M/mL for stickier samples like whole-blood. To be fair, the higher recovery of the PSI (~45%) relative to the sample loops (~25%) means that the samples *should* be diluted a bit more.
2. Periodic Wash solution during runs. I would say at least once an hour, run some Wash solution to help prevent clogs from forming.
3. When clogs form, the majority of them appear to be forming where the silica tube from the PSI unit screws into the grounding nut. So, when your pressure starts going up, unscrew from there, let it drip for a few seconds, then try screwing it back in.
4. Open the Status Panel, and toggle open the Sample Introduction tab. This will allow you to keep an eye on the pressure: when it starts climbing, you can stop sample introduction and deal with the clog before it becomes a major blockage.
5. Here are some guidelines on what the pressure should be:
a) With everything connected and sample running at 30uL/min: no more than about 16psi, usually closer to 14psi. Anything over 18-20psi must be dealt with immediately.
b) With the silica line disconnected from the grounding nut: no more than 4 psi. Anything more than that indicates a clog in the silica line and/or PSI unit.
c) With the silica line disconnected from the bottom of the PSI: no more than about 2psi. Anything more than that indicated a clog in the PSI unit.

Basically, you can forward-flush and backflush the various pieces of tubing, flow sensor, and the grounding nut. The nuts and fittings are standard sizes, so if you have some FPLC or HPLC parts sitting around, you should be able to rig something to flush things with a syringe.


Hope this helps,
Mike

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