CyTOF on Ascite fluid

[Hiranmayi]

Hello,

I've received a request to process ascite fluid from a cancer patient and run immunophenotyping profile to identify potential biomarkers or immune profile of the patient samples using CyTOF. Would someone be able to guide me or point some references in-line to proper method to collect ascite fluid for running cytof, volume of ascite fluid required to capture atleast 1 million events per sample, protocol for processing the fluid for cytof staining/isolation of immune cells for cytof staining etc.

Any help or lead is much appreciated!

Thanks in advance,
Hiran

[mleipold]

Hi Hiran,

This is the only paper I could immediately find where they do CyTOF on Ascites fluid samples: https://doi.org/10.1016/j.omto.2022.03.003
Unfortunately, their experimental isn't very detailed: for example, no mention of what volume was collected per sample.

But, maybe they would share their protocol if you contacted them.


Mike

[Hiranmayi]

HI Mike,

Thank you so much for sharing the link. Will try reaching out to the authors and inquire about the collection protocol.

Best,
Hiran

[rhannan]

Hi Hiran!

My group processes pleural effusions and occasionally ascites. Depending on the px, these fluids can be extremely acellular and the volumes vary wildly. Even with a draw chock full of cells >0.5E6 cells/mL, we still spin down + wash cells prior to counting and resuspension for cryopreservation or immediate staining. If you need to record cellularity of the fluid (always do this!) a final cell count and an initial fluid volume is all you need to back-calculate a concentration. If there was saline used to flush the cavity + ended up in your sample, make sure to account for that exogenous volume (physiologic volume + flush volume = sample volume).

My rule is to process as much volume as possible, or as much as you can get, to maximize the # of cells. If you can get an initial count prior to concentrating, that can guide your decision on how much volume to process.Typically have to do a series of 300xg spins + washes to consolidate the cells into a single tube. RBC lyse if necessary, and then you're off to the races just like you would be with PBMCs. Stain or cryopreserve to your heart's content.

Sadly this work isn't up online, otherwise I'd be happy to link you a paper to reference.


Hope that helps