Plasma versus serum for extracellular vesicle (EV) isolation: A duel for reproducibility and accuracy for CNS‐originating EVs biomarker analysis

Abstract

Blood‐derived extracellular vesicles (EVs) are a popular source of biomarkers for central nervous system (CNS) diseases, but inconsistencies in isolation and analysis hinder their clinical translation. This review summarizes recent studies that investigate the impact of different anticoagulated plasma and serum on the yield, purity, and molecular content of EVs. Specifically, the studies compare ethylenediaminetetraacetic acid (EDTA), citrate, heparin plasma, and serum and highlight the risk of contamination from platelet‐derived EVs. Here, I offer practical guidelines for standardizing EV isolation and analysis, recommending the use of plasma anticoagulated with acid‐citrate‐dextrose (ACD) or citrate followed by EDTA and heparin, subgroup analyses for samples from different biobank repositories, and avoiding serum and plasma‐to‐serum transformation. Other factors like illness, age, gender, meal timing, exercise, circadian timing, and arm pressure during blood draw can alter EV signatures. Yet, how these variables interact with different anticoagulated plasma or serum samples is unclear, necessitating further research. Furthermore, whether the changes are dependent on the isolation or quantification methodology remains an area of investigation. Importantly, the perspective emphasizes the need for consistency in experimental methodologies to improve the reproducibility and clinical applicability of CNS‐originating EV biomarker studies. The proposed guidelines, along with ongoing efforts to standardize blood sample handling and collection, may facilitate the development of more reliable and informative CNS‐originating EV biomarkers for diagnosis, prognosis, and treatment monitoring of CNS diseases.

Analysis of biomarkers in speculative CNS-enriched extracellular vesicles for parkinsonian disorders: a comprehensive systematic review and diagnostic meta-analysis

“…Publication bias analyses showed that smaller studies with non-significant or low effects size results have been less likely to be published. Unsurprisingly, this is to be expected as alluded to previously [ 40 , 41 ], there has been consistent failure of independent validation across studies using speculative CNS-enriched EVs, likely due to EVs being very sensitive to various pre-analytical factors [ 40 ], high complexity of methodologies used to isolate speculative CNS-enriched EVs as well as user differences in handling, among others. Even though measuring biomarkers in speculative CNS-enriched EVs for patients with PD vs HCs has been popular since 2014, only few studies currently exist, further indicating that studies with null results might not have been published.…”

“…As previously indicated [ 40 – 42 ], several pre-analytical elements can have a substantial effect on the purity, content, dimensions, and amount of EVs. Such elements involve the selection of anticoagulation molecules mixed with plasma, EV isolation methodology, the centrifugation procedure, the transportation characteristics, the frequency of freezing and thawing cycles, the storage parameters, the temperature, and the type of tube used for collection.…”

Extracellular Vesicles for Alzheimer’s Disease and Dementia Diagnosis

“…Acknowledging that platelets show circadian rhythmicity, peaking in the morning [153], which in part contributes to platelets having the largest number of EVs in the blood [154], and given the circadian nature of cargoes derived from EVs [155], we considered the timing of collection in our examination of diagnostic accuracy, especially since platelet depletion is commonly overlooked before EV isolation. For both general EVs ( Figure S8A-C ) and speculative CNS-enriched EVs ( Figure S9A-C ), the diagnostic accuracy was consistently higher for studies who obtained their samples in the afternoon vs. the morning ( Table S3 ).…”

“…In a previous review [154], we compared the usage of different anticoagulant molecules on EV isolation and biomarker analysis, and underscored the advantage of using citrate over EDTA and heparin to reduce noise introduced to the biomarker measurements from EVs, which was recently supported by a large comparative study [156]. Our comparative analysis of the diagnostic accuracy using general EVs ( Figure S12A-C ) or speculative CNS-enriched EVs ( Figure S13A-C ) demonstrated that studies using citrate did indeed achieve a higher diagnostic accuracy compared to EDTA ( Table S3 ).…”

Extracellular vesicles from bodily fluids for the accurate diagnosis of Parkinson’s disease and related disorders: A systematic review and meta-analysis

“…As the size, purity, content, and reliability of EVs depends on the liquid and methodology of isolation [100][101][102][103], we compared the diagnostic metrics by CSF, plasma, and serum. We did not include urine or saliva due to the inclusion of only 2 studies for each.…”