Sanford
J Slialtil
Clinical vation
and
pathological
occurs
and
vascular
disorders,
disease,
and
Therefore,
cilitate
identifying
therapeutic
including the
fulfill
migrate
from
the
platelet
platelets
factor
boxane
A2
helpful
(1
and
have
platelet
tiplatelet antibodies were rect detection of activated (2-4).
several
different
conversion
Use
ofthese
receptor, aggregation:
three
sequential
clinical
developed in whole
antibodies
aspects
of the (GP)
several
into
information
phases
by using
antibodies
ligand-binding site within ligand, or for neoepitopes
on
process: a competent
a process required for normal GP lIb-Ilia activation can
posed adhesive receptor-bound
an-
that permit the diblood by flow cy-
provides
lIb-Illa
biochemical
1) adhe-
platelet adhesion be dissected into specific
for the
ex-
GP IIb-IIIa, for the within GP lib or lila
be emphasized. platelet
preparation. to fix blood or after
during
some with
circumparafor-
Under samples
adding
monoclonal
antibody.
rather
than
Second,
is usually
conjugated
chromophore
for
polyclonal
preferred as detection reagents to minimize binding. Third. the activation-dependent to the
First,
activation
bodies are cific-antibody
antinonspeantibody
fluorescein
so that
antibody binding can be quantitated by measuring platelet-associated fluorescein fluorescence in the flow cytometer. When the simultaneous binding oftwo antibodies is assessed, the second antibody is conjugated to biotin, which is detected by flow cytometry after phycoerythrin-streptavidin is added. The use of conjugated antibodies
has
been
before
should
inadvertent
and sample be necessary studies,
maneuvers
collec-
points
to avoid
either
cell activation whole blood,
monoclonal
platelet-activation
maldehyde,
directly secondary
use.
and
drawing it may
of throm-
to sample
physical
surface,
recently platelets
ofglycoprotein
sion and
the
the
reliable released or urine:
markers
related
vein
by yeremain
plasma
metabolites
these
problems limited
1) activated
technical
be taken
whole-blood
is no
to the
Several
must
blood stances fa-
There
The most substances
in the and
understanding
might
care
evaluating
not be activated 3) platelets must
is completed. in vivo are
plasma.
on the basis of circulating acti-
of activation
measuring
at the activated
tometry
and
criteria:
measured
technical
processing
facilitate
changes
and
Although
).
throm-
activation.
must and
4, [3-thromboglobulin,
in research,
To
site
2) platelets manipulation,
stably activated until the assay markers of platelet activation activated
vascular
platelets
following
acti-
in several
peripheral
disorders
platelet
the
platelet
or coronary
of activated
thrombotic
to prevent
that is being sampled, nipuncture or in vitro
from
angina,
to assess thrombotic risk The ideal assay to detect
must
must
that
significance
angioplasty
detection
strategies
platelets
after
certain
platelets
indicate
unstable
and
good in vitro assay platelet dysfunction. vated
studies
is of pathophysiologic
stroke,
bolysis.
tion
thrombotic of using flow
likely
platelets
are
staining
with
ylated
antibodies eliminates the need for adding and for sample washing or centrifugation, to traumatize
platelets
in vitro. Fourth, when a two-color technique discriminated
from
red
an activation-independent
antibody,
flow-cytometer particles: thus,
such
as one
threshold only GP
that
and
induce
platelets is usually and
white
blood
platelet-specific recognizes
GP
microparticles) are analyzed. with a fluorescein-conjugated
dependent
to assess
antibody
activated including
flow cytometry platelets in only cardiopulmonary
cells
by
biotin-
lb. Then,
the
is set to detect phycoerythrin-positive lb-positive particles (that is, platelets
and platelet-derived stained simultaneously
To date,
unwanted
are analyzed in used in which
platelet
Platelets are activation-
activation.
has been
used
to detect
a small number ofclinical bypass and angioplasty
questions must be addressed before the technique wider scale: Can platelets activated in vivo routinely
circulating situations, (2). Several is used on a be detected
exposed during ligand binding; 2) exocytosis-related surface exposure of integral membrane proteins specific for either alpha, dense, or lysosomal platelet granules; 3) surface binding of pro-
by flow cytometry? What is the precise sensitivity and specificity of the flow-cytometric assay compared with existing plasma and urinary assays for platelet activation? Is one activation-dependent
teins, such as thrombospondin, released during platelet secretion; 4) development of platelet procoagulant activity, manifested by the surface expression of binding sites for coagulation factors Va and Villa on both activated platelets and platelet-derived
antibody suming
microparticles Flow whole
.min
(5).
cytometry blood
i Cliii
Nuir
and
‘From 2
can plasma
be
used or after
1992:56:789S-90S.
to study
platelet
separation
Printed in USA.
activation
of platelets
©
in from
1992 American
better than that platelets
Address
the Hospital reprint
pital of the University PA 19104.
another activated
for a particular clinical study? Asin a given disease process enter
of the University requests
to Si Shatill,
of Pennsylvania,
Society for Clinical Nutrition
of Pennsylvania,
Philadelphia.
Hematology-Oncology,
3400 Spruce
Street,
Hos-
Philadelphia,
789S
Downloaded from https://academic.oup.com/ajcn/article-abstract/56/4/789S/4715609 by University of Michigan user on 11 January 2019
Why is platelet activation useful for assessing risk? What are the advantages and limitations cytometry to measure platelet activation?1’2
790S
SHATTIL
the general circulation, how long do these platelets circulate? These questions are being addressed and willhave to be answered before flow cytometry gains widespread use in clinical studies of thrombotic disorders. 13 References
Blood
1990:75:128-38.
5. Sims Pi, Wiedmer T, Esmon of the platelet prothrombinase the platelet plasma membrane. defect in platelet procoagulant 17049-57.
CT, Weiss HJ, Shattil Si. Assembly complex is linked to vesiculation of Studies in Scott syndrome: an isolated activity. I Biol Chem l989;264:
Downloaded from https://academic.oup.com/ajcn/article-abstract/56/4/789S/4715609 by University of Michigan user on 11 January 2019
I. FitzGerald GA, Catella F, Oates IA. Eicosanoid biosynthesis in human cardiovascular disease. Hum Pathol 1987:18:248-52. 2. Abrams CA, Shattil Si. Immunological detection of activated platelets in clinical disorders. Thromb Haemost 199 l;65:467-73.
3. Shattil Si, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987:70:307-15. 4. Abrams CS, Ellison N, Budzynski AZ, Shattil Si. Direct detection of activated platelets and platelet-derived microparticles in humans.