Platelet removal by single-step centrifugation.

The study of extracellular vesicles (EVs) in plasma requires removal of cells including platelets. At present, a two-step centrifugation protocol is recommended and commonly used. A simpler protocol that is less operator dependent is likely to improve the quality of plasma samples collected for EV research. The objective of this study is to develop an easy, fast and clinically applicable centrifugation protocol to produce essentially platelet-free plasma with a high yield for EV research. We compared the two-step centrifugation protocol to a single-step protocol at 5,000 g for 20 minutes. The removal of platelets was computationally predicted and experimentally validated. Flow cytometry was used to detect residual platelets and platelet-derived (CD61+) EVs. The single-step protocol at 5,000 g (i) is less laborious and approximately ten minutes faster, (ii) removes platelets as effective as the two-step centrifugation protocol, and (iii) has a ~ 10% higher plasma yield, whereas (iv) the recovery of platelet-derived EVs is comparable. For future research on plasma EVs we recommend the newly developed, easy and fast single-step protocol for preparation of platelet-free plasma for research on plasma biomarkers including EVs.

Rate zonal centrifugation can partially separate platelets from platelet-derived vesicles.

BACKGROUND: Centrifugation is commonly used as a first step to enrich biomarkers from blood. Biomarkers are separated on the basis of density and/or diameter. However, the centrifugation protocol affects the yield and purity of biomarkers, for example, isolation of platelets results in co-isolation with extracellular vesicles (EVs). OBJECTIVE: To assess the ability of rate zonal centrifugation (RZC) to separate platelets from co-isolated EVs. METHODS: Using a linear Optiprep gradient, RZC was able to separate a mixture of beads with different diameters but similar density. Next, RZC was applied to samples containing both platelets and platelet-derived EVs (n = 3). After RZC, all fractions were collected and stained with anti-CD61-Alexa 488 to measure the concentrations of platelets and platelet-derived EVs by flow cytometry. RESULTS: We confirm that RZC separates polystyrene beads with diameters of 140 nm, 380 nm and 1,000 nm. Next, we show that the majority of platelets occur in fractions 8-19, whereas the majority of platelet-derived EVs are detectable in fractions 1-7. Furthermore, each fraction contains a different diameter range of platelets, which suggests that separation is indeed diameter based. CONCLUSION: RZC can partially separate platelets from EVs.

Detection of extracellular vesicles in plasma and urine of prostate cancer patients by flow cytometry and surface plasmon resonance imaging.

Large (> 1 µm) tumor-derived extracellular vesicles (tdEVs) enriched from the cell fraction of centrifuged whole blood are prognostic in metastatic castration-resistant prostate cancer (mCRPC) patients. However, the highest concentration of tdEVs is expected in the cell-free plasma fraction. In this pilot study, we determine whether mCRPC patients can be discriminated from healthy controls based on detection of tdEVs (< 1µm, EpCAM+) and/or other EVs, in cell-free plasma and/or urine. The presence of marker+ EVs in plasma and urine samples from mCRPC patients (n = 5) and healthy controls (n = 5) was determined by flow cytometry (FCM) and surface plasmon resonance imaging (SPRi) using an antibody panel and lactadherin. For FCM, the concentrations of marker positive (+) particles and EVs (refractive index <1.42) were determined. Only the lactadherin+ particle and EV concentration in plasma measured by FCM differed significantly between patients and controls (p = 0.017). All other markers did not result in signals exceeding the background on both FCM and SPRi, or did not differ significantly between patients and controls. In conclusion, no difference was found between patients and controls based on the detection of tdEVs. For FCM, the measured sample volumes are too small to detect tdEVs. For SPRi, the concentration of tdEVs is probably too low to be detected. Thus, to detect tdEVs in cell-free plasma and/or urine, EV enrichment and/or concentration is required. Furthermore, we recommend testing other markers and/or a combination of markers to discriminate mCRPC patients from healthy controls.

Centrifugation affects the purity of liquid biopsy-based tumor biomarkers.

Biomarkers in the blood of cancer patients include circulating tumor cells (CTCs), tumor-educated platelets (TEPs), tumor-derived extracellular vesicles (tdEVs), EV-associated miRNA (EV-miRNA), and circulating cell-free DNA (ccfDNA). Because the size and density of biomarkers differ, blood is centrifuged to isolate or concentrate the biomarker of interest. Here, we applied a model to estimate the effect of centrifugation on the purity of a biomarker according to published protocols. The model is based on the Stokes equation and was validated using polystyrene beads in buffer and plasma. Next, the model was applied to predict the biomarker behavior during centrifugation. The result was expressed as the recovery of CTCs, TEPs, tdEVs in three size ranges (1-8, 0.2-1, and 0.05-0.2 µm), EV-miRNA, and ccfDNA. Bead recovery was predicted with errors <18%. Most notable cofounders are the 22% contamination of 1-8 µm tdEVs for TEPs and the 8-82% contamination of <1 µm tdEVs for ccfDNA. A Stokes model can predict biomarker behavior in blood. None of the evaluated protocols produces a pure biomarker. Thus, care should be taken in the interpretation of obtained results, as, for example, results from TEPs may originate from co-isolated large tdEVs and ccfDNA may originate from DNA enclosed in <1 µm tdEVs. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.