Surface Profiling of Extracellular Vesicles from Plasma or Ascites Fluid Using DotScan Antibody Microarrays

Abstract

DotScan antibody microarrays were initially developed for the extensive surface profiling of live leukemia and lymphoma cells. DotScan's diagnostic capability was validated with an extensive clinical trial using mononuclear cells from the blood or bone marrow of leukemia or lymphoma patients. DotScan has also been used for the profiling of surface proteins on peripheral blood mononuclear cells (PBMC) from patients with HIV, liver disease, and stable and progressive B-cell chronic lymphocytic leukemia (CLL). Fluorescence multiplexing allowed the simultaneous profiling of cancer cells and leukocytes from disaggregated colorectal and melanoma tumor biopsies after capture on DotScan. In this chapter, we have used DotScan for the surface profiling of extracellular vesicles (EV) recovered from conditioned growth medium of cancer cell lines and the blood of patients with CLL. The detection of captured EV was performed by enhanced chemiluminescence (ECL) using biotinylated antibodies that recognized antigens expressed on the surface of the EV subset of interest. DotScan was also used to profile EV from the blood of healthy individuals and the ascites fluid of ovarian cancer patients. DotScan binding patterns of EV from human plasma and other body fluids may yield diagnostic or prognostic signatures for monitoring the incidence, treatment, and progression of cancers.

Microvesicles in Cancer: Small Size, Large Potential

“…In contrast, Jamaly et al recently used various anticoagulants to isolate MV and measure their total plasma concentrations by NTA without finding any significant differences in numbers [68]. Apart from total MV numbers, the isolation of MV from serum drawn into lithium heparin tubes seems to result in the aggregation of platelet-derived MV, with sticky vesicle pellets and a reduction in platelet MV numbers, thereby specifically affecting certain MV subpopulations [69,70]. In addition to the anticoagulant, other preanalytical factors such as centrifugation, temperature, freeze-thaw cycles, or agitation can influence MV in blood samples, which should be taken into account when setting up isolation protocols for clinical studies [66,67,71].…”

“…Since PPP is often prepared by centrifugation at 2500-3000 × g, this step might result in a significant loss of larger MV, which already pellet at this force [9]. It has been proposed that two centrifugation steps at 1500× g might be preferable for MV isolation [70]; however, more elaborate sorting methods might be required to specifically isolate larger EV from blood.…”

“…Moreover, especially low-abundance tumor MV might be concealed in the heterogeneous mixture of blood MV, which might necessitate the development of novel protocols for specific enrichment of tumor MV populations. One suggestion is to remove the platelet-derived MV prior to analysis [ 70 ] since they represent the largest MV population in blood. A protocol was recently published for isolating different subpopulations of small and large EV from solid tumor tissue, in this example from melanoma [ 122 , 123 ].…”

Phenotypic analysis of extracellular vesicles: a review on the applications of fluorescence

“…Later on, the same group reported the development of an array with capacity of screening 60 surface antigens from blood plasma without prior processing or enrichment [138]. Another established array, initially developed for leukaemia cells (DotScan), has been successfully implemented on vesicles for phenotypic analysis by fluorescence and chemiluminescence-based immunoassays [139].…”

Long-Term Temporal Dynamics of Synaptic Vesicles

“…The current limit for reliable FAVS is ~100 nm (Momen-Heravi et al, 2012). An alternative would be magnetism activated vesicle sorting (MAVS), but this is not currently feasible either, because the size of the magnetic beads necessary for effective separation, which would have to be delivered into releasable vesicles to tag them, exceeds the diameter of the synaptic vesicle (Belov et al, 2016). To assess changes in the molecular composition of synaptic vesicles over time, I thus used a two-colour STED imaging approach on ultrathin slices.…”