CyTOForum

Hello everyone,
I am leading a clinical study utilizing the MDIPA staining protocol from Daniel Geanon et al.'s 2021 study on COVID-19 immune signatures. Our protocol involves initial whole blood staining using the MDIPA kit, followed by fixation with Prot1 reagent, and subsequent freezing at -80°C.
We are facing significant delays from SmartTube Inc., with no confirmation or response to our order inquiries, mirroring experiences of other European labs that typically face a three-month wait.
Considering our project's timeline, I’m looking for alternative reagents that can provide comparable results of Prot1. Does anyone have suggestions for reliable alternatives?
Thanks for any insights you can provide.

You can try Cytodelics. See https://doi.org/10.1016/j.jim.2019.112673
I have not tested it with MDIPA.

Hi Joseena,

I know Petter Brodin's lab (among others) have used it in a number of studies. In the limited testing we've done, Cytodelics vs PROT1 has some minor differences in staining, so probably worth doing a small pilot.


Note: here are a couple papers that do some head to head comparisons of various WB stabilizers (including both PROT1 and Cytodelics) that might help you get started:

1. "Whole blood preservation methods alter chemokine receptor detection in mass cytometry experiments"
Sakkestad ST, Skavland J, Hanevik K
J Immunol Methods. 2019
https://doi.org/10.1016/j.jim.2019.112673
* CyTOF paper

2. "A comprehensive assessment of four whole blood stabilizers for flow-cytometric analysis of leukocyte populations"
https://doi.org/10.1002/cyto.a.24700
* this was a flow paper rather than CyTOF


Mike

Hi Everyone,
Thanks for the insights! I appreciate the heads-up on the comparisons between Cytodelics and PROT1. I'll definitely check out those papers.

Cheers,
Joseena

Dear Joseena,

When you use the MDIPA kit you can freeze the sample after Ir stain directly into the -80. There is an application note for this: https://www.standardbio.com/asset/260
We do not add any preservative buffers after that. We only use those if you do not stain the sample directly upon receiving it.
Is there any specific reason why you would like to add any preservative after the MDIPA stain?

Best wishes,
Marjolijn

I can support this method - we frequently store stained samples in cryopreservation media at -80 until use. If you're feeling adventurous, you can even barcode w the SBT palladium kit after thawing samples

Depending on your volume requirements for Prot1 I may be able to help - please send me a message!

Hi Marjolijn,

Joseena referenced the 2021-Geanon et al COVID paper, which is probably this one: https://doi.org/10.1002/cyto.a.24317

In this case, the workflow was that the collection site would draw blood, do the MDIPA staining, then fix and freeze the MDIPA-stained WB for eventual shipping to a central facility. That central facility would then perform barcoding, intracellular staining, Ir staining, and then run the sample.

This was also the basic protocol for the large NIH COVID19 IMPACC study: 12-14 collection sites around the US would draw the blood, stain with MDIPA, then fix with PROT1 and freeze at -80C. Then, periodically, they would make a large shipment of samples to Stanford or Mt Sinai (6-7 collection sites to Stanford, 6-7 different collection sites to Mt Sinai) who would then do the rest of the experiment.


As such, the protocol was designed to allow MDIPA staining of fresh WB (which allows the chemokine receptors to stain properly), but then allow a stopping point in the protocol after surface stain so that samples could be banked.

I don't know if Joseena's study is doing any intracellular staining or not (or if there's even more than one blood collection site), but banking would still allow them to bank samples until they have a full 20plex amount of samples, to run the samples more efficiently.

So, the application note method of carrying the staining all the way through to Ir before freezing may not be the optimal workflow for them.


One other question: looking at the application note, it states:
"2. Aspirate supernatant leaving ~100 μL residual volume of iridium fix/perm solution.
3. Resuspend the cell pellet in residual volume and transfer into a labeled 1.5 mL microcentrifuge tube.
4. Place 1.5 mL tubes in a plastic container for storage at –80 ºC"

In contrast to Riley's comment about "cryopreservation media" (presumably FBS/DMSO), this application note seems to imply that the sample is frozen at -80C in Ir-Fix-Perm.

Is that correct? If so, I'm surprised, we have not had great success in freezing fully-stained samples in anything other than FBS/DMSO. In particular, we start to see small but noticeable and reproducible losses in Monocytes and B cells in particular.


Mike

Hi Mike,

Thanks for the clarification, I haven't read the paper so my comment was a bit misplaced than, sorry about that.

About the application note;
Yes you do indeed freeze the sample in residual fix/perm with Ir, no FCS/DMSO needed.
Ir does not bind covalently so it will leak out of the cells. When thawing the cells it is nice that you have it in there.
In our hands it works better since you do not have to wash away all the protein in the FCS and the DMSO. Leading to less cells loss during staining procedure.
I just keep it in the 5mL eppendorf tubes which also takes away the extra steps of transferring the cells for freezing and thawing.

About the experience you have with the loss of Monocytes and Bcells, did you add the extra fixation step (freshly made), that FLDM later on added to the surface staining protocol? First mix, do not vortex and then add the fixative (leads to less cell clumps). Also extended vortexing will not be beneficial for these cells as well. But I think you know this better then me

Best,
Marjolijn

Dear Marjolyn,

Thank you for your response.

We are planning to implement the modified MDIPA MaxPar Direct-Smartube whole blood protocol 2 as described in the 2021 study by Geanon et al. Our goal is to adapt this method for our routine clinical lab, which operates from 8 AM to 5 PM, with a focus on reducing sample processing times for samples that arrive late in the day.

Here is a summary of the protocol as adapted from the article:
"300 μl of the MDIPA-stained blood was fixed and stabilized by the addition of 420 μl of Prot1 stabilizer (SmartTube Inc., San Carlos, CA). After a 10-minute incubation at room temperature, the samples were transferred to labeled cryovials and immediately placed in −80°C for long-term storage and/or shipment. According to communications with the manufacturer, the effective concentration of formaldehyde in the Prot1-stabilized samples is greater than 2%, which is expected to effectively neutralize active SARS-CoV-2. Samples were subsequently thawed using the SmartTube Prot 1 Thaw/Erythrocyte Lysis protocol as per the manufacturer's instructions. Afterward, the samples were washed in Cell Staining Buffer and simultaneously fixed with 2.4% PFA in PBS with 0.08% saponin and 125 nM Iridium (Ir) intercalator for 30 minutes at room temperature. Following this, the samples were washed and stored in Cell Staining Buffer (supplemented with 125 nM Ir) until acquisition. To facilitate data acquisition and doublet removal, multiple samples were barcoded using Fluidigm Pd barcoding kits and then washed and pooled for data acquisition."

Best regards,
Joseena

Joseena,

based on your quoted protocol from Geanon etal, and if you remain unable to source enough PROT1, I am confident you will achieve satisfactory results by fixing the MDIPA-stained whole blood in 1-4% PFA and freezing these fixed samples in a 90%FBS 10%DMSO cryopreservation media at -80C. Upon thawing, you can proceed with the Geanon protocol. You will need to sub an alternative RBC lysis reagent - e.g. 0.1% TX-100 - for the SmartTube Erythrocyte Lysis buffer if you also cannot source it.

I am not sure what PROT1 brings to the table beyond some formaldehyde and antifreeze (I believe diethylene glycol), but it is certainly not a 'secret sauce' which cannot be recapitulated in-house.

Riley