If you are using the PBMCs for RNA expression analysis, you can not store them at 4C for more than 2 hours. Since you just need RNA and the drug concentration from the samples and the plasma, I would suggest to use 'RNA later' (a reagent that preserves cellular RNA integrity for gene expression analysis) to store the cells pellet after removing the plasma. The only thing you need to make sure that the RNA Later do not influence the drug concentration analysis. You can find more details about RNA later in storing blood in the attachment.

You can also store the cell pellet directly in -80C and the RNA still be fine if you can take care while thawing and extracting the RNA (Never bring the stored sample above 4C and be quick in processing the sample).

If you want the same same sample for the drug concentration, I would suggest to store the sample in two aliquotes, one for RNA and the other for the drug.

Isolating the PBMC is a laborious procedure if you are collecting the sample at different time points. I hope this information helps. Good luck

The short answer is no. Freezing would interfere with density gradient separation and you would have trouble getting a clean PBMC prep. The best thing would be to keep the blood samples in the fridge overnight, isolate PBMCs/plasma and then freeze the individual components (plasma on its own and PBMCs in RNAlater, as Ramachandramouli suggested). -80 should be fine.

Storing whole blood in the fridge for over a day would not be ideal. Storage at 4 degrees for varying lengths of time may have an impact on gene expression (would depend on the genes you are looking at). I would think you would have to test drug concentrations in the plasma and not in the PBMCs. Also there are special tubes out there which make PBMC isolation easier/faster, if you can afford them (link below).

https://www.youtube.com/watch?v=K9IlBxjgLt4

Thanks both for the replies. Yes I do need to measure drug concentrations in PBMCs as well. Essentially I need to do 1. drug concentration in plasma 2. viral load in plasma 3. drug concentrations in PBMCs 4. gene expression in PBMCs. In one sample and no processing overnight, until the next morning.

So it sounds like there is no way of doing this from what you are saying. I could use a vacutainer CPT tube, but I guess RNA integrity and gene expression might change overnight, even at 4C. I could add RNAlater to a vacutainer CPT tube in advance, but I guess the problem with this would be 1. no longer any vacuum 2. potential microbial contamination which would alter gene expression. From what I can gather there are no vacuntainer CPT (or similar systems) that come with RNAlater or similar RNA preservative. I found an article that suggests RNA is stable at 4C for 24 hours, but not clear if they did comparison to 0 hours and to what extent gene expression was systematically affected (link).

http://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0184692&type=printable

So essentially, I guess there is no way of doing what I want to do.

Sorry, but no.

If you freeze whole blood you will: contaminate the plasma with erythrocyte contents and, load the system with so much protein that it will be very difficult to isolate RNA that means anything.

I'd not count on RNA being *quantitatively* stable for anything like 24 hours. More like 4 if you're lucky.

If it were me, I'd draw the blood into an EDTA vacutainer, and divide it into 2. Spin 1 hard and harvest the plasma. Spin 2 at ~1,000 rpm for 20 mins and harvest the buffy layer. Divide into two. Put 2a into RNA later and freeze 2b for drug content analysis.

Good luck

G

Interesting question.

Following

Thanks. Unfortunately, we can't spin overnight so we wouldn't be able to do that with an EDTA tube.

Thinking about it more though. Why couldn't I add RNALater to an EDTA tube or vacutainer CPT tube (heparin of sodium citrate) in advance? If I did this under sterile conditions, with laminar flow cabinet, sterile needles and syringes etc. Especially if done several weeks in advance of the blood draw. The bung on an EDTA or CPT tube isn't sterile anyway when a blood draw is done.

I would need the tube to still work with a vacutainer needle when it comes to the blood draw, even after puncturing it once to add RNAlater. We want to minimise blood spillage and use of needles when it comes to the blood draw as we are dealing with a biohazard level 4 virus.

Thanks

There's obviously something about this that I'm missing. I don't understand "can't spin overnight". Why can't you harvest the required specimens when you draw the blood...?

Thanks. Because there is no lab overnight. Only nurses, who will do the blood draw, then leave the samples in the fridge. This is in a remote setting in Africa with limited laboratory capacity. The laboratory does not function at night. And because this is a BSL 4 virus, the nurses can't manipulate the samples (in general, but also specifically outside of a lab context). Therefore the samples cannot be processed till the morning.

It's important that we do serial sampling following the first drug dose, for PBMC gene expression and for PK parameters. The timings of the first drug dose is dependent on when the patients are admitted and when they get their first drug dose, which can be at any time. This is not a clinical trial, but an observational study, hence why the timings of the first dose of drug are unpredictable. Unfortunately, it's not feasible to have a lab person in 24 hours a day 7/7.

ok. Thanks.

Then here's what I would do. Harvest the bloods from the overnight as soon as you can, noting the (variable) time since dose and additional (fridge) step. Keep these data separate.

Use the data that you have from the more controlled (daytime?) study as your core data and when you've completed your analysis of them, *then* ask whether the variable time in the refrigerator yielded data that were compatible, and have a statistician tell you whether they can be included.

You've presumably got people coming in with differing viral loads so you have enough variability on your hands as it is without complicating your harvesting procedure. How many patients are you expecting to accrue? Why can't first dose be at a standard time, say 07.00?

Good luck

G

stimulating question and I think the above provided answers are satisfactory

Thanks. The first dose can't be at 7 am as it's not a clinical trial, but an observational study and patients already receive the drug as part of the standard of care. Since many patients are critically ill, the drug is started as soon as they are admitted, whatever the time of day. Unfortunately, our sample size will be small due to limited case numbers, therefore using the data in the manner you suggested probably won't be feasible.