CyTOF XT Performance in comparison to Helios

[LukaTandaric]

Dear Mike,

I am happy to give an update on the contamination situation, albeit a bit late.

CAS+ was used as the suspension medium in both samples (Helios and XT).
After a rigorous acid wash by our FlowCore engineer, the lanthanum and cerium contamination is gone. We are still stumped as to where it originated from. Our only hypothesis is that it came from previously analyzed samples which came from smokers.
As for the barium and lead, they are still present at a constant 10-20 dual counts, but that doesn't seem to noticeably affect other channels.

Concerning the clogging, I have experienced problems with clogging while running fixed whole blood samples (total leukocytes only). Clogging occurred only once while running high-quality samples, but happened repeatedly when running a whole blood sample of not-so-high quality (coagulation occurred due to improper use of EDTA vacutainers, several rounds of filtering and DNAse were done on these samples). The auto-unclogging procedure failed to resolve any of the clogs. We had to pause the machine and switch to a clean nebulizer to continue running.

- Luka

[mleipold]

Hi Luka,

I'm glad that the contamination is gone. By "acid wash", do you mean nitric, Wash (HF), or both?

Yes, samples from smokers can have certain backgrounds (among others, see viewtopic.php?f=10&t=789&p=2319&hilit=mischmetal#p2319 ), though in that case it often includes Neodymium which I don't think you reported.


Regarding clogging of WB samples: Stanford HIMC has just completed our part of the NIH COVID-19 IMPACC study, which used EDTA WB with SmartTube PROT1 buffer fixation. Mt Sinai (the other BLD CYTOF IMPACC site) developed the staining protocol based off their pre-IMPACC COVID studies (eg, https://doi.org/10.1002/cyto.a.24317 ).

In that Cytometry A paper, they mention:
"It is important to note that the standard SmartTube thawing protocol as per the manufacturer's instructions worked well for all healthy donor samples used in our initial validation experiments; however, when applying this protocol to blood collected from hospitalized COVID-19 patients we observed several instances in which the stabilized samples appeared to be partially clotted and exhibited high amounts of debris after thawing and lysis, which we suspect may be related to polymerized fibrin or other plasma factors related to COVID-19 disease-associated coagulopathy. If not addressed, this debris contributed to overall poor sample and staining quality and in some cases precluded analysis of samples. We found that following the red blood cell lysis washes with three additional large volume washes using ~10 ml of PBS + 0.2% BSA with centrifugation at 250 rcf and followed by filtration through a 70 micron filter depleted the majority of this debris and permitted effective analysis of blood samples that would otherwise have been discarded."

Basically, after the ThawLyse steps, there are 3 lower-speed CSB (or CyFACS above) "platelet depletion" washes. It was honestly surprising sometimes how much this can help clean up the sample. It obviously won't un-clot the sample, but the red clotted material seems to remain suspended somewhat at the lower speed (250g rather than 400g) and so it can be aspirated away. So, your cell recovery will still be low (whatever fraction of sample that didn't clot remains), but at least the clumpy material will be greatly decreased and hopefully help your samples run more smoothly.


Mike

[mds4z]

Hello

Has anyone done a direct comparison of the XT to Helios, specifically looking at cell recovery/loss. Our initial test, split sample between two instruments and ran side by side, we seemed to have a significant recovery difference between the two. We had a third less in cell recovery from the XT vs the Helios. Our cell populations percentages and MFI looked equivalent between the two. Just the overall cell recovery. We need to do some more testing, but has anyone else seen this?

Thanks
Mike

[mleipold]

Hi Mike,

If I remember correctly, UVA initially had a CyTOF2 (usually ~25% cell recovery) before you got a Helios (usually ~40% cell recovery).

So, are you finding that the XT cell recover is closer to CyTOF2?


Mike

[ycheng]

Hi all,

We have recently installed a new CyTOF XT, and the recovery was not great, about 40-50%. I usually have 65-80% for Helios if I do everything very carefully. My experience in CyTOF1 and CyTOF2 ranges from 30-45%. My guess is that the automatic sample introduction of XT has complicated loop system, and therefore cause more cell lost. We are thinking if we could manually bypass it for some precious clinical samples.

Yang

[EricHaas]

Has anyone else seen the low Offset values in XT collected data?

I am working on a dataset now that was run on the XT and understand that the offset values typically are relatively lower than Helios values, but has there been any communication on why this is?

Also, has anyone found a way to utilize the amplitude parameter for QC or cleanup? I'd be curious to know!

Thanks,
Eric