Page 100 - Read Online

Lai et al. J Cancer Metastasis Treat 2019;5:65 I http://dx.doi.org/10.20517/2394-4722.2019.011 Page 3 of 9

ADVANTAGE OF PLASMA EV OVER WHOLE PLASMA FOR BIOMARKER DISCOVERY AND
ANALYSIS
EV-based liquid biopsy is essentially a transformation of the traditional assay for biomarkers in plasma or
other bodily fluids into an assay for biomarkers in EVs isolated from either plasma or other bodily fluids.
This transformation is critical as using EVs from bodily fluids as the source of biomarkers as opposed to
unfractionated bodily fluids could greatly enhance the signal to noise ratio in detecting a biomarker. For
example, plasma, a commonly used bodily fluid in diagnosis has a wide dynamic concentration range of
at least 10 orders of magnitude between the most abundant plasma protein, albumin and rare biomarker
[9]
proteins . In fact, > 95% of the protein mass in human plasma is dominated by a dozen proteins with
[10]
albumin and the globulins constituting about 50% and 25%, respectively . As such, disease-associated
protein biomarkers are likely to be rare and masked by the highly abundant proteins, making plasma
biomarker discovery and detection challenging. Specific methods to Isolate EVs from plasma could
potentially remove the highly abundant proteins from the plasma and enhances the discovery and detection
of rare disease-associated biomarkers.

TECHNOLOGIES FOR ISOLATING EVS FROM BODILY FLUIDS
[3]
As extensively discussed in the MISEV2018 , EVs could be isolated from different bodily fluids such as
serum/plasma, urine, tears, milk, spinal fluids using a large variety of techniques. As each of the fluids have
unique features that could interfere with EV purification and analysis, MISEV emphasized the need for
these fluids to be appropriately processed prior to EV isolation and analysis. For example, blood plasma is
routinely treated with anti-clotting agents such as EDTA, citrate or heparin to prevent clotting while the
urine is often depleted of Tamm-Horsfall protein, a major urinary protein. However, MISEV2018 does
not specifically recognise any method as the gold standard for EV isolation but noted that the choice is
dependent on the end use of the EVs. Presently, the most commonly used methods for EV isolation as
described in MISEV2018 are generally based on biophysical parameters such as size (e.g., filtration, size
exclusion chromatography, precipitation), density (e.g., differential ultracentrifugation, sucrose gradient
ultracentrifugation) and charges (e.g., electrophoresis, ion exchange chromatography). EVs are also isolated
according to surface proteins (e.g., immunoaffinity chromatography). Combination of these technologies,
e.g., filtration with density ultracentrifugation are also being used or developed to improve the efficiency,
specificity and purity of the EV preparation.

As noted by several groups, the desired end use of the purified EVs is greatly affected by the choice of
isolation techniques or combination of techniques. For example, precipitation or filtration techniques
could process large volumes of fluids and while this is critical in preparing sufficient therapeutic EVs,
these methods may not be as stringent as size exclusion chromatography in isolating highly enriched EV
preparation [11,12] . As many of the current EV isolation techniques require specialized equipment and many
procedural steps, microfluidic chips are increasingly being used in the isolation of EVs as a means to reduce
the hands-on time and the number of procedural steps. Most of the microfluidic chips to isolate EVs are
also based on the same fractionation parameters as the conventional EV isolation techniques namely
[13]
immune-capture by antibodies against surface proteins of EVs , size fractionation by membranes [14,15] ,
[17]
[16]
[18]
ciliated micropillars , acoustics or viscoelasticity .

Generally, most EV isolation technologies enriched for EVs on the basis of size or densities. Hence most
EV preparations, especially those from biological fluids, are also significantly enriched in similar sized non-
EV complexes such as HDL and LDL lipoprotein complexes and/or protein aggregates, and two or more
isolation techniques often have to be used in tandem to reduce non-EV components [19-23] . In addition, most
of the current EV isolation technologies require relatively large volume of sample fluid, typically 250 µL to
1 mL or long preparation time to isolate sufficient EVs for diagnostic applications.