In Focus
Extracellular vesicles in blood components
Sources of variability and challenges for standardisation
Extracellular vesicles (EVs) are nanoscopic, anucleate structures bound by a phospholipid bilayer, released from nearly every cell in the human body.
EV populations are heterogenous, differing in their physical, chemical and functional properties, depending on the cell of origin and the stimuli inducing release. Historically, EVs were subtyped (e.g. exosomes, microvesicles and apoptotic bodies etc.) based on size, surface markers and/or biogenesis pathway. However, this has led to confusion and contradictions in the published literature. In an effort to standardise EV research, the International Society for Extracellular Vesicles (ISEV) has outlined a set of guidelines (MISEV2023) and criteria to facilitate more consistent nomenclature and characterisation approaches.1 It is recommended that the term EV is used a generic umbrella term, unless specific physical characteristics (e.g. size), biochemical composition (e.g. surface proteins) and cellular origin are defined.
In addition to inconsistencies in nomenclature, differences in starting material, lack of standardised isolation protocols, data acquisition and analysis strategies introduce many sources of variability in EV research. Figure 1 summarises the main areas of concern for EV research in the context of blood components for transfusion.
Figure 1. Sources of variability for extracellular vesicle (EV) research in the context of transfusable blood components.
Blood component manufacture and storage
Blood components can be collected from either a whole blood (WB) donation or via apheresis (Figure 2A and B). As shown in Figure 2A, WB can be leukoreduced, and stored for transfusion (as cold-stored WB), or separated following centrifugation to create red blood cell components (RBCC) and plasma (fresh frozen plasma; FFP). Cryoprecipitate can be made by slowly thawing FFP in the refrigerator, creating a gelatinous precipitate rich in clotting proteins. Leukoreduced platelets (PLTs) and/or plasma can be collected by apheresis, while simultaneously returning the red blood cells back to the donor. PLTs are conventionally stored at room temperature (20-24°C) with agitation. PLTs can also be stored unagitated at 2-6°C (Cold PLTs) or cryopreserved with 5-6% dimethyl sulfoxide (DMSO) and stored below -65°C (Figure 2B). Each component undergoes different collection and manufacture processes, and are uniquely stored for optimal shelf-life, which may differentially affect EV formation. Significant research efforts have been performed using freshly donated peripheral blood, or EVs induced by stimulation with specific activators/agonists. However, comparatively little has been done to identify the EV populations present in blood components, and how these may change as a function of manufacturing methods and storage practises. Further, the clinical significance of EVs in blood components is still unclear, with evidence suggesting they drive important physiological functions (e.g. coagulation) as well as being associated with adverse transfusion reactions.2
EV isolation techniques
Several isolation and purification methods are commonly used to separate EVs from cells, each with their own advantages and disadvantages, and it widely recognised that a combination of techniques may provide the best outcomes.3 Filtration is the simplest method to isolate EVs from a sample through a membrane with a specific permeability. Centrifugation protocols include differential and density gradient based approaches. Differential centrifugation, sequentially separates particles according to size and density, leaving the smaller, lighter particles (i.e. EVs) suspended in the supernatant. Subsequent ultracentrifugation of the supernatant may be performed to collect the EVs for downstream purposes. However, this may also lead to fragmentation of cells and larger EVs. The speed and number of centrifugation cycles may selectively isolate particular EV populations, therefore not providing an accurate representation of the entire population. Size-exclusion chromatography (SEC) separates structures based on their ability to pass through an intricate gel matrix. Depending on matrix porosity and composition, specific EV populations may be selectively eluted. Affinity-based separation techniques are highly specific, as they rely on specific biochemical interactions (e.g. antibody-antigen, ligand-receptor, or peptide-lipid) to selectively isolate EVs from complex biofluids.
Once the supernatant or EV fraction has been isolated, it is typically frozen until characterisation. Storage below -65°C in an ultra-low temperature freezer is recommended for EV preservation, compared to refrigeration, or freezing by liquid nitrogen. However, there is no universally optimised protocol, including the requirement for a cryoprotectant4.
EV characterisation
There are many methods used to characterise EVs (Figure 1), although no single measurement is able to satisfy all EV characterization requirements outlined in the MISEV2023 guidelines.1 Nanoparticle tracking analysis (NTA) is an effective tool to determine the size and quantity of nanoparticles in a sample, but also has the capacity to detect surface biomarkers.5 Flow cytometry and western blotting both utilise antibodies to detect specific biomarkers, although flow cytometry can identify and localise proteins on an individual cell level, rather than the whole sample.6 Transmission electron microscopy (TEM) is highly specialised, but provides crucial insights regarding the size and morphology of EVs, but also an indication of the purity of the sample.7 Functional assays evaluate membrane integrity and bioactivity, and are critical for understanding EV intercellular communication potential and diagnostic or therapeutic potency.8
The heterogeneity of EVs means there is no universal way to define or classify them. As such, a combination of surface-expressed biomarkers is required, for example by identifying the cell of origin, the presence of externalised phosphatidylserine (PS) and tetraspanins (Figure 2C).1, 5 CD235a, recognising glycophorin A, is the most common marker to identify RBC-EVs (Figure 2C), while CD41, CD42 and CD61 have all been used to identify PLT-EVs. Surface-expressed PS is a well-established global EV marker, as it is present on the majority of circulating EVs (Figure 2C), and has functional implications as a signalling molecule, specifically in initiating coagulation. Tetraspanins such as CD9, CD63 and CD81 are highly abundant in EV membranes, reportedly being up to 7- to 124-fold more abundant in EVs than their cell of origin.9
Conclusion
Extracellular vesicles represent a complex and biologically important component of blood products, with growing recognition of their relevance to transfusion medicine. However, progress in understanding their functional significance has been hindered by inconsistent nomenclature, variable isolation procedures, and diverse characterisation methodologies.
Future research should prioritise harmonised methodologies for EV isolation and characterisation, alongside comprehensive evaluation of EV phenotype and function across different blood products. Improved standardisation and deeper mechanistic understanding will be essential to determine the role of EVs in blood component quality, transfusion efficacy, and the development of novel diagnostic and therapeutic applications.
Figure 2. Blood component collection and storage methods, and a phenotypic approach to characterise extracellular vesicles (EVs) present in these components.
A. WB can be leuko-reduced, and stored for transfusion, or separated by centrifugation to create a red blood cell concentrate (RBCC) and plasma (fresh frozen plasma; FFP)
B. An apheresis machine can selectively collect platelets (PLTs) while simultaneously returning the red blood cells and most plasma back to the donor. PLTs are leukoreduced and stored on an agitator at room temperature (20-24°C; RT PLTs). PLTs can be stored unagitated at 2-6°C (Cold PLTs) or cryopreserved with 5-6% dimethyl sulfoxide (DMSO) and stored below -65°C (Cryo-PLTs).
C. A simplified schematic of an approach to characterise RBC-EVs and PLT-EVs, by interrogating externalised phosphatidylserine (PS), transmembrane tetraspanins (i.e. CD9 and CD81), and a cell of origin marker (CD235a, CD61). Illustrations are made using BioRender.
References
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