A beginner's guide to study extracellular vesicles in human blood plasma and serum

Abstract

Blood is the most commonly used body fluid for obtaining and studying extracellular vesicles (EVs). While blood is a standard choice for clinical analysis, using blood as a source of EVs introduces multiple layers of complexity. At the Blood Extracellular Vesicle Workshop organized by the International Society for Extracellular Vesicles in Helsinki (2022), it became evident that beginner researchers lack trustworthy information on how to initiate their research and avoid common pitfalls. This educational guide explains the composition and frequently used terminology of blood, provides guidelines for blood collection, and the preparation of plasma and serum. It also introduces the basic principles of isolating and detecting blood EVs while considering blood-related factors. The goal of this guide is to assist beginners by offering a concise and evidence-based introduction to the current knowledge and available resources to study blood EVs.

Keywords: blood; detection; exosomes; extracellular vesicles; guidelines; isolation; plasma; review; serum; vesicles.

FIGURE 1

Fractionation of blood by centrifugation. The collected blood is centrifuged to separate cells from the blood fluid. Centrifugation separates blood into three distinct fractions. The red bottom fraction contains predominantly erythrocytes. The size of this fraction is approximately 40%–50%, but this depends on the donor. The intermediate thin white layer, also known as ‘buffy coat’, constitutes less than 1% of the total volume and contains leukocytes and most of the platelets. The leukocyte population comprises the peripheral blood mononuclear cells, such as monocytes, T‐ and B‐lymphocytes, as well as polymorphonuclear cells like neutrophils, and the rarer cell types, basophils and eosinophils. The yellow top fraction is the blood plasma, which primarily consists of water (90%–92%), and the rest of this fraction is comprised of soluble proteins, lipoproteins, residual platelets, fragments of other cells such as ‘ghosts’ from red blood cells, and EVs. Image created by Biorender.com.

FIGURE 2

Preparation of plasma and serum. Plasma (top, from left to right) is prepared from anticoagulated blood by centrifugation. The protocol shown is an example, but this protocol is commonly used by dedicated blood EV laboratories. In this protocol, blood is first centrifuged at 2500×g for 15 min to separate the blood cells from the plasma. Centrifugation is performed at 20°C using a swing‐out rotor and without applying a break (cold activates platelets and a fixed angle rotor introduces more platelets/material to the side of the tube, which may become loose when spinning is stopped). During centrifugation, the blood separates into three clearly distinct fractions, as explained in Figure 1. Next, the plasma is carefully collected by pipetting while leaving approximately 0.5–1 cm safety margin on top of the buffy coat. This safety margin aims to minimize the re‐entry of platelets (predominantly present in the loose buffy‐coat) into the collected plasma. Next, the plasma is then transferred to a new tube and subjected to another centrifugation step using the same conditions as before. The final plasma sample is collected without disturbing the cell pellet, which is best achieved by leaving another 0.5–1 cm from the bottom if possible. It is important to maintain a constant safety margin between samples and measure the platelet count in the final prepared plasma. Finally, plasma can be used directly, and/or stored at −80°C in single‐use aliquots with suitable volumes for the planned downstream analyses to avoid multiple freeze‐thaw cycles. Serum (bottom, from left to right). Similar to plasma, all serum samples should be treated identically, including standardizing the time the blood is allowed to clot on the bench, typically 30–60 min. There are various types of commercial serum tubes available, but glass tubes without any additives also suffice. At present, optimized serum preparation protocols for EV research have not yet been established. Therefore, we recommend using the same centrifugation protocol as for plasma preparation after the clotting step. Image created by Biorender.com.

FIGURE 3

Coagulation pathways and platelet activation. Coagulation can be initiated through two different routes: the intrinsic and extrinsic pathways. The intrinsic pathway of coagulation (top left) is triggered by an activating surface. A well‐known example of intrinsic coagulation pathway initiation is contact activation by glass, which activates factor XII to factor XIIa. Glass tubes were commonly used for blood collection in the past. In contrast, in a physiological setting, blood can contact extravascular tissue in a bleeding wound, which results in the activation of the extrinsic pathway of coagulation (top right). This pathway begins with the transmembrane protein tissue factor (TF), which acts as a receptor for coagulation factor VII (present in the blood), and this (extrinsic tenase) complex results in the formation of (activated) factor VIIa. Both the intrinsic and extrinsic pathways converge into a final common pathway. In the common pathway, factor X is activated into factor Xa, and factors Xa and factor Va together form the prothrombinase complex, which catalyses thrombin formation (IIa) from prothrombin (II). The formation of tenase and prothrombinase complexes require a surface, which is present as membranes (platelets, EVs) that expose negatively charged phospholipids (PL) as phosphatidylserine. Several coagulation factors (II, VII, IX, X) require Ca²⁺ ions for binding to the procoagulant membrane. Certain anticoagulants inhibit coagulation by chelating Ca²⁺ ions thereby impeding the formation of the tenase and prothrombinase complexes. Thrombin also activates platelets (right), leading to the secretion of granule contents, platelet aggregation, and the release of EVs. In turn, platelet‐derived EVs promote coagulation by generating additional procoagulant (coagulation‐promoting) PL membrane surface. It is important to note that this is a highly simplified overview, and the coagulation pathway is interconnected to the fibrinolytic pathway and complement activation. From the regulatory perspective, the coagulation process is actively counter‐counterbalanced by natural anticoagulants in blood and on the vascular wall. When preparing serum, spontaneously generated thrombin triggers fibrin formation and activates platelets, which explains the higher concentration of platelet‐derived EVs in serum compared to plasma prepared from the blood of the same donor (as shown in Figure 4). Image created by Biorender.com.

FIGURE 4

Concentrations of blood cell‐derived extracellular vesicles in plasma and serum. Blood was collected with informed consent from a single healthy donor in accordance with the rules of the Medical Ethical Committee of the Amsterdam Medical Centre, University of Amsterdam (W19_271#19.421). Venous blood was collected using a 21‐Gauge needle, and the first 2 mL of blood was discarded. To prepare plasma and serum, blood was centrifuged for 15 min at 2500 g and 20°C, without using a brake. After centrifugation, the tube was marked 1 cm above the cells using a Lego brick. The supernatant was carefully collected using a plastic Pasteur pipet to 1 cm above the cells, and pipetted into a new tube. This new tube containing either plasma or serum was centrifuged as described for the first centrifugation step, and then the (supernatant) plasma and serum were collected to determine the concentrations of EVs and platelets as described earlier (van der Pol et al., 2012). Blood was collected into four different blood collection tubes. One tube type contained the anticoagulant EDTA for plasma preparation (Becton & Dickenson ((BD), New Jersey, US; 368861), while the other three tubes were used for serum preparation: A (serum CAT, activator silica; BD; 368815), B (serum SSTII, activator silica; BD; 367955), and C (serum, no activator, Greiner Bio‐one, Kremsmünster, Austria; 455001). Multiple tubes of EDTA plasma were collected and prepared either immediately after blood collection (arbitrarily denoted as t = 0) or after the tubes had been left standing on the lab bench for 1 h or 3 h (t = 1 or t = 3). Similarly, the different serum blood collection tubes (which require time to clot, hence no t = 0) were also left on the bench and centrifuged after 1 h or 3 h. The figure presents the concentrations of the following extracellular vesicles (EVs): (A) leukocyte‐derived EVs; CD45+), (B) platelet‐derived EVs (CD61+), and (C) erythrocyte‐derived EVs (CD235a+), measured by calibrated Apogee A 60 enabling measurement of EVs within the size range of 160–1000 nm. Measuring EVs and platelets by flow cytometry was performed as described (van der Pol et al., ; Zhang et al., 2021). This figure demonstrates that (1) the concentrations, particularly of platelet‐derived EVs, differ between plasma and serum, and (2) differences in pre‐analytics of serum preparation (such as the choice of collection tube, the time between blood collection and centrifugation) also impact the measured concentrations of EVs.