It is very important in a study the size of MaGiCAD that every sample
is prepared in exactly the same way. In order to achieve this, detailed
protocols have been prepared that clearly indicate the way in which
a sample should be treated. The most important of these are given
Summary of blood fraction preparation
From each patient involved in MaGiCAD, blood is taken from the arterial
sheath. For the majority of the patients the sheath is placed in
the femoral artery, although in approximately 2% of cases the sheath
is placed in the brachial or radial arteries. Approximately 40 ml
of blood is withdrawn into a 50 ml syringe, and platelet-poor
plasma and serum prepared as follows.
The highest quality platelet-poor plasma (ppp) is made with a
minimum of platelet degranulation. In order to reduce platelet
degranulation to its minimum, Diatube H CTAD tubes (Beckton-Dickinson)
are used, which contain 3.2% citrate together with a mixture of
platelet degranulation inhibitors. The Diatube H tubes are pre-cooled
on ice. Immediately upon receiving the syringe of blood 4.5 ml
of whole blood is added to each diatube, and the tube inverted
and replaced on ice. The remainder of the blood is used to make
Following incubation on ice for between 15 and 60 minutes, the
diatubes are spun at 2500g at 4°C in a pre-cooled centrifuge.
The blood cells are pelletted, and only 1 ml of the plasma from
the centre of the supernatant is taken and frozen in aliquots at
The remaining blood in the syringe is put into 15 ml polypropylene
tubes and left to clot at room temperature. After clotting for between
2 and 3 hours the tube is spun at 4000g for 5 minutes. Usually the
clot does not readily spin down on the first attempt. In these cases,
the clot is loosened from the wall of the tube and spun down again.
Once the clot is spun down to leave the serum above as a supernatant,
the serum is removed and transferred into a fresh 15 ml tube. The
serum is spun once more to pellet all remaining red blood cells,
and the supernatant transferred into the 1 ml microtitre plates.
All samples are stored at -80°C in 96 well plates that have
a maximum capacity of 1 ml.
All of the materials mentioned above are detailed in the table below: