FAQ  •  Register  •  Login

Barcoding questions

<<

dtelad11

Master

Posts: 129

Joined: Mon Oct 31, 2016 6:26 pm

Post Sun Aug 25, 2019 2:48 pm

Barcoding questions

I'm preparing a community survey regarding barcoding usage with the CyTOF and could use some background about a few of the technical elements of barcoding:

1) If I recall correctly, barcoding allows more effective use of some reagents. I am guessing that this means the antibody cocktail; did I get it right? Are there other reagents that can be used more effectively by pooling samples? How much does barcoding save in this context? (Either % of reagent or estimated dollar amount?)
2) Does barcoding save on machine run time? I think it is, since the operator doesn't need to switch between tubes. If yes, how much time is saved? (Again, estimated % time is totally fine.)
3) What are some of the reasons for NOT barcoding? I have "it costs money", "I don't know how to do it", "I don't have enough samples for barcoding", "I can't fix my samples", any other ideas?
<<

mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Mon Aug 26, 2019 2:58 pm

Re: Barcoding questions

Hi El-ad,

In my experience:
1. You *do* often save some reagent usage: in many cases, we were able to cut the antibody amount in half. For example, for 10 samples barcoded together, this means we could use 5 tests rather than 10 tests.
However, this did *not* apply in every case. And, the barcode agents (antibody or small molecule) do have a cost themselves: in our hands, using the Fluidigm barcodes, the amount of antibody reagent staining that we saved was basically offset by the cost of the kits. So, the total cost per debarcoded sample basically wound up the same.

2. The main way barcoding saves on machine time is by not having to switch samples. I'd say this saves you 10% on time? Imagine 20min acquisition for a normal sample, then 2min or so involving MilliQ rinse (to minimize carryover) and then getting the next sample going. You can also save a bit of time running Wash solution periodically: if you run Wash at the end of each BC'd sample, you can often time it so that you're there when the target time/event number is hit. Then, you can run Wash while the data processing finishes: this usually takes a couple minutes at least, so you're not losing time running Wash during that.

3. If you can't fix your samples, you can't really run them on CyTOF: even if you're not staining intracellularly (not even Ir), the MilliQ washes will bust your cells. I guess this isn't an issue if you're running with CAS, but we don't.

The main issue with barcoding is that one bad sub-sample will poison the entire combined BC'd sample. This could be due to:
a) Sample clumping. One bad sample clumps the entire BC'd sample, causing you to take a *major* hit on cell recovery (as well as overall data quality, since clumpy samples don't stain or wash efficiently).
b) Poor viability. Dead cells often soak up reagent, which can throw off your effective titer to cause dimmer staining.
c) Debris. This often goes with poor viability, but remember, debris from one sample will cause streaking in the *entire* BC'd sample. This streaking will therefore be raised background in *every* debarcoded sample.

I've had instances of all 3.

d) Somewhat separate: very few people do the "exhaustive" Bodenmiller-like barcoding that maximizes the total number of barcodes. However, if they do, and you have a BC reagent failure, you can *miscode* and confuse your samples.....worst case scenario, miscode a PMA+I as an Unstim.


Barcoding is great, but it is *not* a magic wand.


Mike
<<

dtelad11

Master

Posts: 129

Joined: Mon Oct 31, 2016 6:26 pm

Post Tue Aug 27, 2019 6:57 pm

Re: Barcoding questions

Thanks Mike! Excellent points and exactly the information I needed.

Wholeheartedly agree that barcoding is not magic. I promise to post to the forum as soon as I find the magic reagent that makes all biology experiments easy ;)
<<

mleipold

Guru

Posts: 5792

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Aug 27, 2019 7:56 pm

Re: Barcoding questions

Hi El-ad,

One more thing regarding "why I don't barcode".

One concern that a number of people who do ICS assays have is focused on the rare but non-zero cross-contamination you can have after debarcoding (depending on thresholds, etc).


Specifically, imagine that you have 3 conditions:
1. Unstim
2. Peptide stim (antigen-specific, so rare but present)
3. PMA+I (hits a large number of cells)

The concern I've heard from some of my ICS colleagues is usually focused around "how can I be sure that the Cytokine-positive cell events I get in my debarcoded Peptide stim *aren't* PMA+I events that slipped through the debarcoding?" This may be less of an issue when the number of Antigen-specific cells is higher (eg, CMV) than others (eg, Peanut, which may be in the 5-50 per 10M CD4+), but I've had more than one researcher raise this question.

One way that some people have dealt with this is by having an Unstim combined sample, a Peptide combined sample, and an PMA+I combined sample; or Unstim+Peptide combined sample and then a PMA+I sample.


I'm curious on the Cytoforum users' thoughts on this:
1. How many people are concerned about this in ICS assays?
2. If you are, do you avoid it by just running individual samples and not barcoding?
3. Or do you group similar samples (eg, all Unstims) together rather than all total samples together?
4. Or are you just super strict on your thresholds, and accept the greater hit to overall recovery?


Mike
<<

sedejong

Participant

Posts: 7

Joined: Wed Mar 04, 2015 3:42 pm

Post Wed Aug 28, 2019 8:07 pm

Re: Barcoding questions

Regarding the problem of imperfect debarcoding of samples with for example ICS, I think a similar issue is also present when you do not barcode, but then due to imperfect washing.

When I was testing barcoding, I barcoded separate samples with the Fluidigm kit and ran them separately on the CyTOF. I washed between the samples (10 min wash, 10 min nitric acid, 10 min milliQ), but I could still see carry over from one sample in the other. Although I must say that the cells that carried over where mostly positive for highly expressed markers such as CD3 and the other metals seemed to be washed away. So if you do a quick wash (with only milliQ) between samples that are not barcoded, I think the risk of carry over will be larger and more difficult to recognize.

I guess it is a choice between two evils and all dependent on how strict your are with debarcoding or how long you want to wash...
<<

AdeebR

Grand master

Posts: 169

Joined: Thu Mar 13, 2014 5:58 pm

Location: NYC

Post Tue Sep 03, 2019 8:04 pm

Re: Barcoding questions

I completely agree with Sanne's point regarding the potential risk of cross sample carryover when running sequential (non barcoded) samples, and like the fact that barcoding can at least give you some indication of whether a suspect cell reflects cross sample contamination.

When performing any barcoding experiment where the focus is on subsets that may be rare in some samples and abundant in others, I think it's very important not to treat the debarcoding threshold as a standard "set it and forget it" parameter. My current preferred workflow is to use the Zunder lab debarcoder that encodes the barcode separation and Mahalanobis distance as parameters in the FCS file. In addition to allowing more accurate custom tailoring of the barcode separation for each sample, this also allows you to go back and double check the barcode separation of specific cells at various stages of your analysis, e.g. on those rare cells that you're worried may reflect cross-sample contamination. If they show clear barcode separation then I can be pretty confident that they originated from the sample I think they did, whereas if the separation is poor I'd be more concerned that they may have "slipped through" from another sample.
Adeeb Rahman
Icahn School of Medicine at Mount Sinai, NYC

Return to CyTOF general discussion

Who is online

Users browsing this forum: No registered users and 14 guests