Hi everyone, Ken Witwer here from Johns Hopkins University. My part of the MOOC is to speak with you about pre-processing of blood plasma and serum. We'll actually mostly be talking about blood plasma today. So, I'm recording this talk on a Sunday afternoon in Baltimore, Maryland in the United States and for those of you who know Baltimore, you know that Sunday is dirt bike day. So that's when the dirt bikes take to the streets and they're actually quite loud so if you hear a noise from outside my office that doesn't sound like me, that's probably a dirt bike and as much fun as those dirt bikers are having out there today. I think we're going to have just as much fun talking about plasma and serum. So blood is of course, a very common extracellular vesicle source in our biological studies. Every biofluid that we have examined contains EVs this is not a surprise because cells are constantly secreting and releasing EVs into their surroundings. So any biofluid that is exposed to cells should contain EV's. However, of all of the EV-containing biofluids, blood products, such as serum and plasma, are the most commonly used biofluids in EV studies. There are many reasons for this. Some of the reasons include the relative homogeneity of these fluids after they had been processed, the ease of access to blood, so the concept of the liquid biopsy mostly involves blood. And compared with other biofluids blood is the most easily accessed with a few exceptions such as urine or saliva. Finally, we have extensive archives of blood samples from well conducted clinical studies that go back decades in many cases. Should you use serum or plasma in your EV studies? This is a very good and important question. Over here on the right, we see a serum collection tube where the straw colored serum is separated from the blood cells by a gel. So this tube can be used to obtain blood and then to centrifuge it such that we are left with a population of serum. Serum is the fluid that is recovered after clotting in the collection tube. It's important to note that serum is not a physiological fluid, except in wound healing. We should also note the EVs may be slightly more abundant in serum than in plasma. Why is this? It's because many serum EVs are released by platelets during the coagulation process that leads to the production of serum. Here are a few citations. Some citations from the 1980s and 1990s that helped to establish these concepts. What about plasma? So a few years ago when we were submitting the first physician paper of the International Society for Extracellular Vesicles, one of our reviewers had a very eloquent way of describing plasma, and that was, as the physiological fluid of the blood that occurs within the circulation. Of course, to maintain this fluid, once the blood is obtained, we must add an anticoagulant to prevent clotting. We also must be careful to remove cells. And these would include both nucleate and anucleate cells, especially the platelets. Protein content of plasma may influence depletion of certain particles and some then treat their plasma with thrombin. So, the idea of thrombin treatment is to convert fibrinogen to fibrin. You polymerize that and remove the fibrin and this will result in a serum-like product. However, it's important to keep in mind that thrombin is also a platelet agonist. So by removing protein content of your plasma you may also be changing some other properties of that plasma. When would it be appropriate to use serum? There's certainly many cases in which serum might be the appropriate fluid of choice. One of these is if the study that you wish to examine has collected only serum, so there are many clinical studies where serum might be available in archive and plasma might not. There are also of course, biological questions that would involve platelets where you would want to enrich for platelet derived EVs or you might wish to enrich for platelet derived or platelet nucleic acids. You would wish to keep in mind, however, that many of the EVs in those serum samples may have been released after the blood draw. When would you use plasma, the International Society for Extracellular Vesicles recommends plasma as the preferred fluid for most blood EV studies and that is again because plasma is the physiological fluid of blood. However, we need to be careful that we have standardized handling that would include the removal of cells and platelets to allow within and between study comparisons. So we want to talk about the pre-processing steps in generating plasma for EV studies. And of course, pre-processing could mean any step up to the final analysis, or that final experiment that you want to do with your EVs. But for our purposes in this particular move, you just want to talk about pre-processing as the technical steps that are occurring before the EV isolation. So some general considerations in obtaining blood would include the following. Discard the first tube or the first few mills of blood. So keep in mind that the act of obtaining blood is actually a traumatic process. You're forcing a metal tube, a hollow metal tube into someone's arm, in most cases. And so you're going to be generating some activated cells and some trauma products that would include fibroblasts. You don't want to be examining these cells or the products of these cells. Secondly, process the blood quickly. It's best if you can process the blood within 30 minutes of draw. But the earlier the better, so I think that these are concepts that very few would disagree with. Next, the right anticoagulant must be chosen. There are two main categories of anticoagulant. The first is the chelating agents such as, EDTA, the EDTA based formulations, oxalate, and the citrate based formulations including, sodium citrate, ACD, and CTAD. The second category is the protease inhibitors. And the most widely used of these, of course, is heparin. And heparin has been used in many, many clinical investigations. The problem with heparin is that it can interfere with downstream assays like PCR. So if heparin is present within your plasma, additional purification steps are needed in order to obtain PCR results that are reliable. Heparin can also alter the cellular EV uptake and release ratio. So, if heparin is present while your cells are still in the plasma, heparin is going to effect how EVs are taken up or released from those cells. Some had also reported effects of EDTA on EV uptake and release but others have not. Oxalate and citrate have similar actions as EDTA. We recommend that you use citrate-containing anticoagulants in your EV studies. However, they may be room for more investigations into how EDTA and oxalate could be effective anticoagulants in the EV studies. Beyond the anticoagulant what are some steps that you can take to avoid platelet activation, so the same sort of platelet activation that occurs in the generation of serum. How can we avoid this in our plasma studies? One possibility is to use a needle gauge as large as comfortable for your donor, and over here on the left we depict several different needles ranging from the sub-q needle on the bottom to an 18 gauge needle on the top, and you can see the difference in their bore. The idea here is that reducing the turbulence within the needle will also reduce platelet activation. And so some have recommended large needles and also the avoidance of butterfly setups to avoid platelet activation. However, the evidence here is actually somewhat ambiguous. A second consideration is to remove the tourniquet when blood flow begins. Again, you're going to reduce pressure and thereby reduce activation of platelets. Thirdly, do not shake the blood. So once the blood has been obtained, you want to invert it gently to make sure that the blood and the anticoagulant are mixed. But you'll then keep the tube upright and not expose it to a lot jostling. Finally, do not chill the blood. This is a mistake that is often made. There are times when blood should be chilled. But in EV studies at least in most EV studies, this is not appropriate. So you don't want to store the blood on ice or in the cold room. And in fact until the platelets are completely removed, you do not want the samples to drop below room temperature. Removing cells and platelets from plasma. You now have your blood. You've taken pains not to mishandle it. What do you do next? The first thing to do is to make sure that you remove the blood cells, so the red blood cells, the white blood cells you want to generate something that's known as platelet rich plasma. This can be done by centrifuging the sample at 300 to approximately 800 RCF for 10 minutes. Following this, two successive spins will be done at 2500 xg. These steps are recommended by the International Society for Thrombosis and Hemostasis and also by ISO to remove most of the platelets in a plasma sample. These spins will be conducted at 2500 xg for 10 to 15 minutes each. After each spin, most of the plasma will be taken from the tube and transferred into a new tube. You want to make sure to maintain at least a little bit of plasma at the bottom of these tubes so that pellets are not disrupted. Importantly, record all of the details of these steps. So what centrifuge did you use? What kind of a tube did you use? What was the column volume? What was the column height? Remember to record all of the details of your procedure so that others can follow it when they read your report in the literature. Following these spins the samples may be stored at minus 80 degrees Celsius or at higher temperatures depending on how long the plasma is meant to be stored. Depending on the type of EV that you wish to study, you might wish to go onto a higher speed spin before storage. Now that we've talked about some of the important steps in plasma pre-processing, let's talk now about substandard archive samples. The past is gone, and can you do anything with a group of samples that you're able to obtain that's been in the freezer for a while. First of all, you want to be aware of the different sample pre-processing steps that may have been done within the same study. These are very important study design questions. Let's take an example of where you have a set of disease samples that are very valuable, you've gotten them from a collaborators clinic. And you want to know how do I compare these with healthy donor samples, or some other appropriate control. You want to find out exactly what the protocols, the supplies, every detail that was associated with that plasma preprocessing. What those were in the case of these samples? Then who are the donors if you have a control population? Do you have match cases in controls or how well were these cases and controls matched. Based upon the results of this analysis, are you able to proceed with your study? In other words, can you answer your question or do you need to have a new control population? So you want to know what the sample processing was. You want to be able to evaluate it. And you want to be able to report the differences to allow appropriate interpretation of your results. What happens when you have a population, a steady population where you know that the plasma has been obtained through pre-processing steps that you would not necessarily use if you were in control of that study? Can improperly, or perhaps differently archived samples be used? Now in the case of controls, if controls are missing or inadequate you can always get new controls. And you can obtain these new controls by matching or following the case protocols. You would want to remember to do freeze-thaw steps and other manipulations to mimic the original handling, the handling of those original samples, the disease samples presumably. There's also some evidence that all is not lost if samples have not been processed the way that you might prefer. So Manish Tiwari's group has published recently on how plasma samples can possibly be remediated after they have been collected by careful centrifugation protocols. So I give this citation here. Please consult this. In other words, pre-processing conditions are very important for plasma EV studies. However, there may be some approaches that will help you to remediate samples that were obtained even without these best practices. I would point to two studies, or two papers for further reading and these include a report from the ISDH and a report from ISO. Thanks everyone.
