Subject: Prot/Smart Tube Sample Integrity Issues and Request

[singh423]

Subject: Prot/Smart Tube Sample Integrity Issues and Request for Advice
Dear CyTOF Team,
We have recently encountered significant issues with the integrity of our Prot Tube/Smart Tube whole blood samples due to fluctuations in the -80°C freezer temperature. The samples turned from pinkish-red to a darker blackish-red, which I believe may be a result of temperature-induced blood clotting, red blood cell rupture/lysis, and damage to the fixed white blood cells. This has led to resistance to lysis, hindering the retrieval of WBCs from the tubes.
Upon microscopic examination, we observed very little or no evidence of lymphocytes. Additionally, after staining the cells with lineage antibodies (CD3, CD19, and CD14), we did not detect any signals for these markers.
If anyone has experienced a similar situation or has suggestions on how to retrieve healthy immune cells from these Smart Tubes, we would greatly appreciate your advice.
Best regards,
Amar

[mleipold]

Hi Amar,

Unfortunately, in my experience, if PROT1 WB samples go dark and clumpy, they cannot be rescued and must be thrown out. Typically, the *entire* sample becomes a single sticky "clot" that cannot make it up a P1000 pipet tip, and cannot be dissolved by the ThawLyse solution. In *most* cases, this is due to improper temperature (or unstable temperature). I have also encountered cases where this has happened if the ratio of PROT1 solution to WB was insufficient.

I have also encountered samples that were dark where the clotting did not occur. In these cases, the samples were overfixed (substantially beyond the 10min RT incubation), and the RBCs could not be lysed even in MilliQ water. As a result, almost all the cells recovered on the CyTOF were RBCs, which we were not interested in. Therefore, again, those samples had to be tossed for our project.


In some "slightly" clumpy cases, you may be able to pipet it (not one giant clot, but something sort of like red "snowflakes"). If that happens, then you can at least try the lower-spin washes mentioned here: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013522/
"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."

In the IMPACC COVID-19 study, we adapted this slightly to do 3x4mL CSB washes (250xg) in 48well 5mL deepwell blocks, and did rescue some of the slightly clumped samples.


If anyone has any pointers on how to rescue dark and fully-clotted/clumpy PROT1 samples, I think there are a lot of people (including myself!) who would like to know. But I haven't heard of any successes......


Mike