Hi, I am Priscila and I am a Scientific Content Writer at Izon Science.
In this video I will briefly go through tips and practical suggestions to Isolate EVs from blood.
Blood is an incredibly resourceful biofluid for EV research,
as blood EVs come from different cellular origins,
representing multitude of physiological states.
Blood is the one of the most used liquid biopsies, as is minimally invasive for the subject.
However, blood is a complex matrix that can have substantial pre-analytical variability.
This is due in large part to the fact that blood cells,
particularly platelets,
can be activated easily and release EVs during sample collection and processing.
One limitation about working with blood samples,
is that the amount of biofluid obtained from one donor,
as blood collection tubes hold a maximum of 10 mls and 50% of blood corresponds to plasma/serum.
Another limitation can be that blood collection requires a phlebotomist,
a person certified to withdraw blood,
however specialized lab facilities are not necessary.
Therefore, a few considerations are necessary to preserve the
physiological status of different blood-derived EVs and minimise their artificial modifications.
In this workflow, the process starts when blood sample is retrieved from test subject.
Then plasma or serum is obtained from blood by removing cellular components,
leading to a light-yellow clear liquid sample.
An optional but commonly used storage step follows,
which helps to maintain the integrity and biology of EVs until full isolation protocol can be continued.
After an appropriate EV purification step such as size exclusion chromatography qEV columns,
an optional post-concentration step can follow to finally yield a pure EV enriched preparation.
The first consideration is how much blood volume will I need.
As mentioned before,
half the volume of blood corresponds to plasma or serum,
therefore for every 10 mls of blood,
5 ml of plasma or serum will be obtained.
In general,
depending on the EV isolation method,
there is approximately 1 or 2 times 10 to the nine EVs/mL of plasma or serum.
Another important distinction is the sample of interest, which can be plasma or serum.
Mainly,
plasma is the cell-free liquid portion of blood when clotting is prevented,
and serum is the cell-free liquid portion of blood when clotting is allowed.
How is coagulation prevented?
With the presence of anticoagulants in the blood collection tube used to withdraw blood.
There are more than ten different blood collection tubes available in market to choose from,
containing different anticoagulants or other components,
most times colour coded for easy visualisation.
The choice of anticoagulant in the tube may have significant effects in downstream analyses,
as for example,
heparin and EDTA residues my interfere with PCR reactions.
How long can blood stay in tube?
Studies have shown that EVs significantly increase within 3 hours of unprocessed blood samples.
Therefore, it is recommended to process blood under
one hour as well as minimise agitation during transport of blood sample.
Like other EV-containing samples, cells must be gently removed by centrifugation at 200 g for 20 minutes.
At this stage, is important to have no brakes in the centrifugation step to avoid damage of cells.
Platelets are the smallest of blood cells,
they have functions on the clotting process,
and they must be removed with slightly higher speed centrifugations.
Thus,
once blood is in the appropriate tube,
a portion of platelets can be eliminated with gentle centrifugation at 400 g for 15 minutes at room temperature,
resulting in a sample called Platelet Rich Plasma.
Next, Platelet poor plasma can be obtained by centrifuging sample at 1,550 x g for 20 minutes at RT.
Finally, platelet Free plasma can be obtained by two spins at 2,500 x g for 15 min at RT.
The importance of platelet removal lies on the fact that platelets normally activate by injury signals from blood vessels in the body,
but they can also activate by shear or cold.
Upon activation,
platelets generate significant amount of EVs,
therefore, it is critical to avoid exposure of platelets in the plasma/serum
sample to cold temperature and remove them as quickly as possible,
before they artificially populate the sample with their EVs during the handling process.
As mentioned before,
ideally, samples should be freshly obtained
and immediately processed to achieve maximal EV recovery and stability.
However, most of time full EV purification
is not feasible after plasma/serum samples are obtained,
requiring some storage.
The two variables investigated to maintain EV stability have been storage temperature and time.
Some studies have helped determining effects on specific EV populations such as nanoEVs and microEVs,
however there is still some debate.
Data shows that plasma stored at -20 ⁰C or -80C maintains nanoEV numbers for months/years of storage,
as opposed to storage of plasma at RT or 4C,
in which nanoEVs increase through time,
most likely due to artificial generation of EVs from residual platelets.
In the same way,
studies performed in microEVs have shown that storage at 80C increase EVs number in Platelet Poor Plasma,
possibly due to platelet activation and release of microEVs.
The first step into EV isolation is the optional separation of microEVs by centrifuging plasma/serum
sample at 10,000 or 20,000 g for 30 minutes to pellet microEVs and resuspending them in desired buffer volume.
The supernatant is then ready for purification by size exclusion chromatography qEV columns.
There are two types of resins in qEV columns,
one has a pore size of 35 nanometres
and isolation particle range of 35 to 350 nanometres
and the other has a pore size of 70 nanometres and isolation particle range of 70 to 1000 nanometres.
The particle size ranges in these columns overlap,
but also emphasize on isolation of EVs from two big groups,
exosomes or microvesicles.
There are different qEV columns suitable for different sample input volumes,
however the preferred qEV among researchers for plasma samples are qEV2,
since they seem to match the plasma or serum volumes obtained from one person’s blood sample.
qEV columns ca be run with the Automatic Fraction Collector (AFC),
with automated flushing and collection of fractions and customisable settings,
like buffer volume and amount and volume of each fraction.
The AFC allows standardization and reproducibility on the EV purification process.
Here is an example of fraction profiling with a qEV2, where particles counts are shown in bars and protein in lines.
There are 3 or 4 EV-rich fractions, which are separate form most of the proteins present in plasma/serum.
After the qEV run,
EV-rich fractions can be pooled and used as such
or can be further concentrated for any downstream applications that require highly
enriched EV samples in small volumes.
For this post-concentration step, most common method seems to be ultrafiltration.
Ultrafiltration allows a separation of EVs by size,
by passing the sample through a filter with protein-based pore sizes,
for example 10, 100 or 300 kDa.
Everything larger than that pore size is retained, basically all EVs, whilst most proteins pass through.
However, there is also a new product launched by Izon, which enables concentration of EVs after purification with qEV.
This qEV concentration kit is based on polymer particles which bind to EVs with high affinity enabling pelleting
the whole complex without the need of ultracentrifugation speeds,
which might damage integrity of EVs.
Finally, sample characterisation can be performed with EV count,
size and charge assessments by TRPS,
molecular profiling can be done by quantification of DNA,
RNA and proteins present in EVs;
presence of population-specific EV markers can be done by western blot assays;
or visualisation done by microscopy techniques.
Thank you for watching and please visit www.izon.com
for application notes and guides to help you work with different types of nanoparticle samples..