Journal of Pathophysiology of Haemostasis and Thrombosis
Evaluation Of Platelet Function By Flow
Cytometry
Alan D. Michelson, M.D
Center for Platelet Function Studies, Departments of Pediatrics, Medicine, and Pathology, University of Massachusetts Medical
School, Worcester, MA, U.S.A.
Key Words
Platelets Flow cytometry
Introduction
Flow cytometry, a remarkably versatile tool for the
study of platelet function, encompasses multiple assays for
multiple purposes (Table 1).
Flow cytometry rapidly measures the specific charac-
teristics of a large number of individual cells. Before flow
cytometric analysis, single cells in suspension are fluores-
cently labeled, typically with a fluorescently conjugated
monoclonal antibody. In the flow cytometer, the sus-
pended cells pass through a flow chamber and, at a rate of
up to 2000 cells per second, through the focused beam of a
laser. After the laser light activates the fluorophore at the
excitation wavelength, detectors process the emitted fluo-
rescence and light scattering properties of each cell. The
intensity of the emitted light is directly proportional to the
antigen density or the characteristics of the cell being
measured.
Alan D. Michelson, M.D., Director, Center for Platelet Function Studies, Room S5-846, 55 Lake Avenue North, Worcester, MA 01655, U.S.A. Telephone: +1-508-856-0056.
FAX: +1-508-856-4282. e-mail: michelson@platelets.org.
Clinical studies that utilize flow cytometric assays of
washed platelets or platelet-rich plasma are, like other as-
says of platelet function, potentially susceptible to artifac-
tual in vitro platelet as a result of the obligatory separation
procedures. The introduction of whole blood flow cytome-
try by Shattil et al.1 was therefore a major step towards the
application of flow cytometry to clinical settings. A typi-
cal schema of sample preparation for whole blood flow cy-
tometric analysis of platelets is shown in Table 2. The an-
ticoagulant is usually buffered sodium citrate, although
other anticoagulants can be used.2 The purpose of the ini-
tial dilution is to minimize the formation of platelet aggre-
gates.2 A minimum of 2 monoclonal antibodies is used,
each conjugated with a different fluorophore. A wide vari-
ety of fluorophores are available for antibody conjugation
(e.g. phycoerythrin, fluorescein, peridinin chlorophyll pro-
tein [PerCP], phycoerythrin-Cy5, phycoerythrin-Texas Red
[RED-670], allophycocyanin [APC]). The test mono-
clonal antibody (recognizing the antigen to be measured) is
added at a saturating concentration. The "platelet identi-
fier" monoclonal antibody (e.g. glycoprotein [GP] Ib-,
GPIX-, integrin IIb-, or integrin 3-specific) is added at a
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near saturating concentration. Physiological agonists can
be used in the assay, including thrombin, thrombin recep-
tor-activating peptide (TRAP), adenosine diphosphate
(ADP), collagen, the complement fraction C5b-9, and
thromboxane A2 analogs. Non-physiologic agonists in-
clude phorbol myristate acetate and calcium ionophore
A23187. Samples are stabilized by fixation, typically with
a final concentration of 1% paraformaldehyde. The anti-
bodies can be added after fixation, provided fixation does
not interfere with antibody binding.2,3 Samples are then
analyzed in a flow cytometer. After identification of plate-
lets both by their characteristic light scatter and by (for ex-
ample) phycoerythrin positivity, binding of the (for exam-
ple) fluorescein isothiocyanate (FITC)-conjugated test
monoclonal antibody is determined by analyzing 5,000 -
10,000 individual platelets. Refs. 4,5 contain specific meth-
odological protocols of whole blood flow cytometric as-
says of platelet function, together with a discussion of
methodological issues.
There are many advantages to flow cytometric analysis of
platelet function. Platelets are directly analyzed in their
physiological milieu of whole blood (including red cells
and white cells, both of which affect platelet activation6,7).
The minimal manipulation of the samples prevents artifac-
tual in vitro activation and potential loss of platelet sub-
populations.1,8-10 Both the activation state of circulating
platelets and the reactivity of circulating platelets can be
determined. The flow cytometric method permits the de-
tection of a spectrum of specific activation-dependent
modifications in the platelet surface membrane. Further-
more, as new monoclonal antibodies directed against novel
functional epitopes are developed, they can easily be in-
corporated into the assay. A subpopulation of as few as
1% partially activated platelets can be detected by whole
blood flow cytometry.10,11 Only minuscule volumes (~5
L) of blood are required1,8 making whole blood flow cy-
tometry particularly advantageous for neonatal studies.12
The platelets of patients with profound thrombocytopenia
can also be accurately analyzed. Finally, flow cytometric
evaluation of platelets is easily adaptable to animal studies
provided the antibody reagents are available.13-15
There are some disadvantages to flow cytometric
analysis of platelet function. First, flow cytometers are ex-
pensive instruments to purchase and maintain. Second, for
a clinical assay, sample preparation can be quite compli-
cated, although the development of kits (e.g. BioCytex,
Marseilles, France) has simplified some of the assays.
Third, to avoid ex vivo platelet activation, blood samples
should be processed within approximately 30 minutes of
drawing for many assays.1 For the evaluation of some
platelet receptors, this time issue can be circumvented by
immediate fixation.2
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Measurement of Platelet Activation
In the absence of an added exogenous platelet agonist,
whole blood flow cytometry can determine the activation
state of circulating platelets, as judged by the binding of an
activation-dependent monoclonal antibody. In addition to
this assessment of platelet function in vivo, inclusion of an
exogenous agonist in the assay enables analysis of the reac-
tivity of circulating platelets in vitro. In the latter applica-
tion, whole blood flow cytometry is a physiological assay
of platelet function in that an agonist results in a specific
functional response by the platelets: a change in the surface
expression of a physiological receptor (or other antigen or
bound ligand), as determined by a change in the binding of
a monoclonal antibody. In addition, as discussed below,
whole blood flow cytometric enumeration of monocyte-
platelet aggregates and procoagulant platelet-derived mi-
croparticles are also sensitive markers of in vivo platelet
activation.
Markers of Platelet Activation
1. Activation-Dependent Monoclonal Antibodies
Laboratory markers of platelet activation include acti-
vation-dependent conformational changes in integrin
IIb3 (the GPIIb-IIIa complex, CD41/CD61), exposure
of granule membrane proteins, platelet surface binding of
secreted platelet proteins, and development of a procoagu-
lant surface (Table 3). The two most widely studied types
of activation-dependent monoclonal antibodies are those
directed against conformational changes in IIb3 and
those directed against granule membrane proteins.
Integrin IIb3 is a receptor for fibrinogen and von
Willebrand factor that is essential for platelet aggrega-
tion.16 Whereas most monoclonal antibodies directed
against IIb3 bind to resting platelets, monoclonal anti-
body PAC1 is directed against the fibrinogen binding site
exposed by a conformational change in IIb3 of activated
platelets (Table 3).17 Thus, PAC1 only binds to activated
platelets, not to resting platelets. Other IIb3-specific ac-
tivation-dependent monoclonal antibodies are directed
against either ligand-induced conformational changes in
IIb3 (ligand-induced binding sites, LIBS)18 or receptor-
induced conformational changes in the bound ligand (fi-
brinogen) (receptor-induced binding sites, RIBS)19 (Table
3). Rather than IIb3-specific monoclonal antibodies,
fluorescein-conjugated fibrinogen can also be used in flow
cytometric assays to detect the activated form of platelet
surface IIb3,20,21 but the concentration of unlabeled
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plasma fibrinogen and unlabeled fibrinogen released from
platelet granules must also be considered in these assays.
The most widely studied type of activation-dependent
monoclonal antibodies directed against granule membrane
proteins are P-selectin (CD62P)-specific. P-selectin is a
component of the granule membrane of resting platelets
that is only expressed on the platelet surface membrane af-
ter granule secretion.22 Therefore a P-selectin-specific
monoclonal antibody only binds to degranulated platelets,
not to resting platelets. The activation-dependent increase
in platelet surface P-selectin is not reversible over time in
vitro.23,24 However, in vivo circulating degranulated plate-
lets rapidly lose their surface P-selectin, but continue to
circulate and function.14,25 Platelet surface P-selectin is
therefore not an ideal marker for the detection of circulat-
ing degranulated platelets, unless a) the blood sample is
drawn immediately distal to the site of platelet activation,
b) the blood sample is drawn within 5 minutes of the acti-
vating stimulus, or c) there is continuous activation of
platelets. The length of time that other activation-
dependent surface markers remain expressed on the platelet
surface in vivo has not yet been definitively determined.
2. Leukocyte-Platelet Aggregates
P-selectin mediates the initial adhesion of activated
platelets to monocytes and neutrophils via the P-selectin
glycoprotein ligand 1 (PSGL-1) counter-receptor on the
leukocyte surface.22 Monocyte-platelet and neutrophil-
platelet aggregates are readily identified by whole blood
flow cytometry (Figure 1).
Fig.1. Whole blood flow cytometric analysis of monocyte-platelet aggre-
gates and neutrophil-platelet aggregates in a normal donor after activation with
thrombin receptor activating peptide (TRAP) 20 M. Monocytes and neutrophils
were identified by their characteristic light scatter properties and the binding of
FITC-conjugated CD14-specific monoclonal antibody TUK4 (left panel). Platelet-
positive monocytes (i.e. monocyte-platelet aggregates) were identified by the bind-
ing of the phycoerythrin (PE)-conjugated IIb (CD41)-specific monoclonal antibody
5B12 in the monocyte region (upper right panel). Platelet-positive neutrophils (i.e.
neutrophil-platelet aggregates) were identified by the binding of the PE-conjugated
IIb (CD41)-specific monoclonal antibody 5B12 in the neutrophil region (lower
right panel). SSC-H, side scatter height
Tracking of autologous infused biotinylated platelets in
baboons by three color whole blood flow cytometry en-
abled us26 to directly demonstrate in vivo (Figure 2) that: 1)
platelets degranulated by thrombin very rapidly (within 1
minute) form circulating aggregates with monocytes and
neutrophils; 2) the percent of monocytes with adherent in-
fused platelets is greater than the percent of neutrophils
with adherent infused platelets; and 3) the in vivo half-life
of detectable circulating monocyte-platelet aggregates (ap-
proximately 30 minutes) is longer than both the in vivo
half-life of neutrophil-platelet aggregates (approximately 5
minutes) and the previously reported14 rapid loss of surface
P-selectin from non-aggregated infused platelets.
All these findings suggested that measurement of circu-
lating monocyte-platelet aggregates may be a more sensi-
tive indicator of in vivo platelet activation than either circu-
lating neutrophil-platelet aggregates or circulating P-
selectin-positive non-aggregated platelets. We therefore
performed 2 clinical studies in patients with acute coronary
syndromes.26 First, after percutaneous coronary interven-
tion (PCI), there was an increased number of circulating
monocyte-platelet (and, to a lesser extent, neutrophil-
platelet) aggregates, but not P-selectin-positive platelets, in
peripheral blood. Second, of patients presenting to an
Emergency Department with chest pain, patients with acute
myocardial infarction had more circulating monocyte-
platelet aggregates than patients without acute myocardial
infarction and normal controls. However, circulating P-
selectin-positive platelets were not increased in chest pain
patients with or without acute myocardial infarction.26
In summary, we have demonstrated by five independ-
ent means (in vivo tracking of activated platelets in ba-
boons [Figure 2, upper panel],26 human PCI,26 human
acute myocardial infarction,26 stable coronary artery dis-
ease27 [discussed in Section II.B.1 below], and human
chronic venous insufficiency28 [discussed in Section II.B.3.
below]) that circulating monocyte-platelet aggregates are a
more sensitive marker of in vivo platelet activation than
platelet surface P-selectin.
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Fig. 2. Baboons were infused with autologous, biotinylated platelets that were (upper panel) or were not (lower panel) thrombin-activated pre-infusion. Surface P-selectin on the
infused platelets and participation of the infused platelets in circulating monocyte-platelet and neutrophil-platelet aggregates was determined by 3-color whole blood flow cytometric
analysis of peripheral blood samples drawn at the indicated time points. The 0 time point refers to blood samples taken immediately pre-infusion. Platelet surface P-selectin is ex-
pressed as mean fluorescence intensity (MFI), as a percentage of the fluorescence of a pre-infusion maximally activated (10 U/mL) thrombin control sample. Monocyte-platelet and
neutrophil-platelet aggregates are expressed as the percent of all monocytes and neutrophils with adherent infused platelets. Data are mean S.E.M.. Reproduced with permission
from ref. 26

3. Platelet-Derived Microparticles 5. Shed Blood

As determined by flow cytometry, in vitro activation of
platelets by some agonists (e.g. C5b-9, collagen/thrombin,
and the calcium ionophore A23187) in the presence of ex-
tracellular calcium ions results in platelet-derived mi-
croparticles (defined by low forward angle light scatter and
binding of a platelet-specific monoclonal antibody) that are
procoagulant (determined by binding of monoclonal anti-
bodies to activated factors V or VIII or by annexin V).29-31
These findings suggest that procoagulant platelet-derived
microparticles may have an important role in the assembly
of the "tenase" and "prothrombinase" components of the
coagulation system in vivo. A flow cytometric method for
the direct detection of procoagulant platelet-derived mi-
croparticles in whole blood has been developed.32
Because of the minuscule volumes of blood required,
whole blood flow cytometry can be used to analyze the
shed blood that emerges from a standardized bleeding time
wound.8,34-36 The time-dependent increase in the platelet
surface expression of P-selectin in this shed blood reflects
in vivo activation of platelets.8,34-36 Immediate fixation
(prior to antibody incubation) is obligatory, in order to ob-
serve these time-dependent changes. The assay can be
used to demonstrate deficient platelet reactivity in response
to an in vivo wound, for example during cardiopulmonary
bypass.35 In addition, by tracking of infused platelets with
biotin or PKH2 (see Section IX.A below), shed blood can
be used to detect the functional participation of the infused
platelets in in vivo platelet aggregates.144

4. Platelet-Platelet Aggregates
Platelet-platelet aggregates can be measured by flow
cytometry on the basis of light scattering properties. How-
ever, if the platelets are aggregated, the amount of antigen
per platelet cannot be determined by flow cytometry.14,33
This is because flow cytometry measures the amount of
fluorescence per individual particle, irrespective of whether
the particle is a single platelet or an aggregate of an un-
known number of platelets. However, a rough estimate of
aggregate size can be made by analyzing the increased
platelet-specific fluorescence.
Platelet Activation in Clinical Disorders
1. Acute Coronary Syndromes
Platelets play an important role in the pathogenesis of
coronary artery disease, including unstable angina and
acute myocardial infarction.37 Whole blood flow cytomet-
ric studies have demonstrated circulating activated plate-
lets, as determined by activation-dependent monoclonal an-
tibodies, in patients with stable angina, unstable angina,
and acute myocardial infarction.27,38-40 In addition, as de-
termined by activation-dependent monoclonal antibodies,

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PCI results in platelet activation in coronary sinus
blood.41,42
Flow cytometric analysis of platelet activation-
dependent markers can be used to determine optimal anti-
platelet therapy in clinical settings, e.g. in acute coronary
syndromes43,44 and after coronary stenting.45,46 Flow cy-
tometric analysis of platelet activation markers before PCI
can predict an increased risk of acute and subacute
ischemic events after PCI.47-50 Flow cytometrically de-
tected exposure of LIBS is strongly associated with the de-
velopment and progression of heart transplant vasculopa-
thy.51
The PlA2 polymorphism of GPIIIa has been reported to
be associated with ischemic coronary syndromes.52 Flow
cytometry has been used to demonstrate that a) PlA2-
positive platelets display a lower threshold for activation,
and b) platelets heterozygous for PlA alleles show increased
sensitivity to antiplatelet drugs.53
Circulating leukocyte-platelet aggregates are increased
in stable coronary artery disease,27,53 unstable angina,38
acute myocardial infarction,26,54-56 and cardiopulmonary
bypass.57 Circulating leukocyte-platelet aggregates also
increase after PCI,26 with a greater magnitude in patients
experiencing late clinical events.58 As discussed in Section
II.A.2 above, circulating monocyte-platelet aggregates (but
not neutrophil-platelet aggregates) are a more sensitive
marker of in vivo platelet activation than platelet surface P-
selectin in the clinical settings of stable coronary artery
disease,27 PCI,26 and acute myocardial infarction.26 Fur-
thermore, circulating monocyte-platelet aggregates are an
early marker of acute myocardial infarction.56
Platelet-derived microparticles are increased in acute
coronary syndromes59 and cardiopulmonary bypass.34,60
2. Cerebrovascular Ischemia
Platelets play an important role in the pathogenesis of
ischemic cerebrovascular disease.61 Increased circulating
P-selectin-positive, CD63-positive, activated IIb3-
positive platelets, platelet-derived microparticles, and
monocyte-platelet aggregates have been reported in acute
cerebrovascular ischemia.62-68 This platelet activation is
evident 3 months after the acute event, suggesting the pos-
sibility of an underlying prothrombotic state.64,66-69 Fur-
thermore, increased expression of surface P-selectin on
platelets is a risk factor for silent cerebral infarction in pa-
tients with atrial fibrillation.70
Platelet-derived microparticles are increased after tran-
sient ischemic attacks.34,71 Increased platelet-derived mi-
croparticles and procoagulant activity occur in sympto-
matic patients with prosthetic heart valves and provided a
potential pathophysiological explanation of cerebrovascu-
lar events in this patient group.72
Enhanced systemic platelet degranulation is associated
with progression of intima-media thickness of the common
carotid artery disease in patients with,73 or without,74 type 2
diabetes (as determined by platelet surface CD63 and
CD40 ligand, and by platelet surface P-selectin, respec-
tively).
3. Peripheral Vascular Disease
Circulating activated platelets and platelet hyperreactiv-
ity (as determined by P-selectin expression, platelet aggre-
gates, and platelet-derived microparticle formation) are in-
creased in patients with peripheral arterial disease com-
pared with healthy volunteers.75,76 Circulating monocyte-
and neutrophil-platelet aggregates are significantly greater
in the early postoperative period in patients with peripheral
vascular disease who go on to develop later graft occlu-
sion.77
With regard to peripheral venous disease, Peyton et
al.28 demonstrated an increased presence of monocyte-
platelet aggregates in the lower extremity veins of patients
with chronic venous stasis ulceration, as compared to con-
trol individuals without venous disease. Interestingly,
these changes were present not only in blood drawn from
the lower extremity veins of affected individuals, but also
in blood drawn from arm veins, suggesting that the
changes are systemic rather than localized to the lower ex-
tremities.28 Powell et al.78 further characterized these find-
ings as being related to the presence of chronic venous dis-
ease rather than the presence of venous ulceration, since
increased numbers of monocyte-platelet aggregates were
noted in patients with all classes of venous disease, not just
in patients with deep venous valvular insufficiency. Fur-
thermore, increased levels of monocyte-platelet aggregates
were noted even in patients with only superficial venous
stasis disease manifested by the presence of varicose veins.
Even more intriguing is the fact that the number of mono-
cyte-platelet aggregates remains elevated six weeks after
total correction of the venous insufficiency by stripping of
the abnormal veins, leaving normal venous physiology as
documented by postoperative duplex scanning.79 This
finding suggests an underlying predisposition to the devel-
opment of chronic venous disease in these patients, perhaps
mediated by monocyte-platelet interactions.
4. Other Clinical Disorders Associated with Platelet Hyper-
reactivity and/or Circulating Activated Platelets
There are numerous other conditions in which whole
blood flow cytometric measurement of platelet hyperreac-
tivity, circulating activated platelets, and/or circulating
leukocyte-platelet aggregates may prove to have a clinical
role, including diabetes mellitus,73,80-82 cystic fibrosis,83
pre-eclampsia,84,85 placental insufficiency,86 migraine,87
nephrotic syndrome,88 hemodialysis,89 sickle cell disease,90
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systemic inflammatory response syndrome,91 septic multi-
ple organ dysfunction syndrome,92,93 antiphospholipid syn-
drome,94 systemic lupus erythematosus,94 rheumatoid ar-
thritis,94,95 inflammatory bowel disease,96 myeloprolifera-
tive disorders,97,98 and Alzheimer disease.99 High levels of
circulating monocyte-platelet aggregates can predict rejec-
tion episodes after orthotopic liver transplantation.100
Uremic patients with thrombotic events have higher num-
bers of circulating platelet-derived microparticles than
those without thrombotic events.101
Reduced Circulating Activated Platelets and Platelet
Hyporeactivity
In addition to the detection of increased circulating ac-
tivated platelets and platelet hyperreactivity discussed in
Sections II.B above, whole blood flow cytometry may be
useful in the clinical assessment of reduced circulating ac-
tivated platelets and platelet hyporeactivity although
there are few published studies in this area.
1. Very Low Birth Weight Preterm Neonates
Compared to adults, the platelets of very low birth
weight preterm neonates are markedly hyporeactive to
thrombin, ADP/epinephrine and thromboxane A2, as de-
termined by flow cytometric detection of a) the exposure of
the fibrinogen binding site on IIb3, b) fibrinogen bind-
ing, c) the increase in platelet surface P-selectin, and d) the
decrease in platelet surface GPIb.12 Furthermore, proco-
agulant platelet-derived microparticles are decreased in
preterm neonates compared with adults.32 This platelet hy-
poreactivity of preterm neonates, as judged by both activa-
tion-dependent platelet surface changes and the generation
of procoagulant platelet-derived microparticles, may con-
tribute to the propensity of very low birth weight neonates
to intraventricular hemorrhage.102
2. Acute Myeloid Leukemia
Prediction of hemorrhage in thrombocytopenic patients
has been problematic because of the lack of laboratory
markers or validated clinical assessment tools.103 The
clinical decision to prophylactically transfuse platelets is
therefore usually based solely on the platelet count.104
However, low levels of expression of platelet surface P-
selectin, as determined by whole blood flow cytometry,
have recently been reported to be a prognostic marker for
hemorrhage in acute myeloid leukemia.103
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Diagnosis of Specific Disorders
Platelet Surface Glycoprotein Deficiencies
1. Bernard-Soulier Syndrome
Bernard-Soulier syndrome is an inherited deficiency of
the GPIb-IX-V complex.105 Flow cytometric analysis with
GPIb-, GPIX-, and GPV-specific monoclonal antibodies
provides a rapid and simple means for the diagnosis of the
homozygous and heterozygous states of Bernard-Soulier
syndrome.106 Whole blood flow cytometry allows analysis
of platelets without attempting the technically difficult pro-
cedure of physically separating the giant Bernard-Soulier
syndrome platelets from similarly-sized red and white
blood cells. Because light scatter (especially forward light
scatter) correlates with platelet size, light scatter gates may
need to be adjusted in the flow cytometric analysis of giant
platelet syndromes such as Bernard-Soulier syndrome.
This adjustment may result in overlap of the light scatter of
giant platelets with red and white blood cells. It is there-
fore essential to include in the assay a platelet-specific
monoclonal antibody as a platelet identifier. For Bernard-
Soulier syndrome platelets, this identifier antibody obvi-
ously cannot be GPIb-, GPIX-, or GPV-specific.
2. Glanzmann Thrombasthenia
Glanzmann thrombasthenia is an inherited deficiency
of integrin IIb3.105 Flow cytometric analysis with IIb-
and 3-specific monoclonal antibodies provides a rapid
and simple means for the diagnosis of the homozygous and
heterozygous states of Glanzmann thrombasthenia.106,107
In addition, a panel of activation-dependent monoclonal
antibodies can be used to evaluate patients with defects in
platelet aggregation,108 secretion,109 or procoagulant activ-
ity.110
Storage Pool Disease
Inherited dense granule storage pool deficiency, a rela-
tively common cause of a mild hemorrhagic diathesis, can-
not be reliably diagnosed by standard platelet aggregome-
try.111 The conventional method to establish the diagnosis
of storage pool disease is to label the platelets with the
fluorescent dye mepacrine and then measure platelet fluo-
rescence by microscopy.112 This assay is based on the se-
lective binding of mepacrine to adenine nucleotides in
dense granules. The assay is not ideal for the clinical labo-
ratory because it is subjective, tedious, and examines only
a small number of platelets. In contrast, dense granule
storage pool deficiency can be accurately diagnosed by a
simple, rapid, one-step flow cytometric assay in a clinical
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laboratory.113,114 The method shows good correlation with
fluorescent light microscopic methods, but improves the
detection of the mepacrine-loaded platelets by quantita-
tively measuring fluorescence on a large number (5000) of
platelets113,114 Acquired dense granule storage pool defi-
ciency, which occurs in myeloproliferative disorders and
end-stage renal failure, can also be diagnosed by flow cy-
tometry.113,115 An alternative approach to the flow cy-
tometric diagnosis of storage pool disease is to measure in-
traplatelet serotonin (5-hydroxytryptamine) with a sero-
tonin-specific monoclonal antibody.116
Heparin-Induced Thrombocytopenia
Unlike normal sera and sera from patients with quinine-
or quinidine-induced thrombocytopenia, sera from patients
with heparin-induced thrombocytopenia (HIT) generate
procoagulant platelet-derived microparticles from normal
platelets.117 This observation has been used to develop a
rapid, specific, and sensitive flow cytometric assay for the
diagnosis of HIT.118 Alternative flow cytometric ap-
proaches to the diagnosis of HIT have been described.119
Monitoring of Antiplatelet Agents
The in vivo effect of the thienopyridines (clopidogrel
and ticlopidine) on platelet function can be monitored by
the vasodilator-stimulated phosphoprotein (VASP) assay
(BioCytex, Marseilles, France).120,121 Through the use of
arachidonic acid as the agonist, flow cytometry can also be
used to monitor aspirin therapy.122,123
Flow cytometric methods can be used to monitor
GPIIb-IIIa antagonists (abciximab, eptifibatide and tirofi-
ban) by measuring receptor occupancy by these drugs.
These methods can be categorized as either direct or indi-
rect. One type of direct method is a competitive binding
assay with either: a) biotinylated124 or FITC-conjugated125
GPIIb-IIIa antagonists; b) blocking monoclonal antibod-
ies;126,127 c) peptides with very high affinities for GPIIb-
IIIa (disintegrins128 or cyclic RGD peptides129); or, d) fi-
brinogen binding after platelet activation (detected by a
polyclonal anti-fibrinogen antibody).130 Another type of
direct method measures the binding of an antibody directed
against the GPIIb-IIIa antagonist.131,132 Indirect methods
measure either: a) the GPIIb-IIIa antagonist-induced bind-
ing of an anti-LIBS antibody;133 or, b) platelet aggregation,
as determined by light scatter.124
Monitoring of Thrombopoiesis
Whole blood flow cytometric methods have been de-
veloped for the identification of young platelets (i.e. those
containing mRNA) by their staining with thiazole or-
ange.134-136 Because of the analogy to reticulocytes, these
thiazole orange-positive platelets have been termed "reticu-
lated platelets"137 and have been used to monitor throm-
bopoiesis.135 Thrombocytopenic patients whose bone mar-
row contains normal or increased numbers of megakaryo-
cytes have significantly elevated proportions of circulating
reticulated platelets.134 In contrast, the proportion of re-
ticulated platelets in thrombocytopenic patients with im-
paired platelet production (reduced bone marrow megakar-
yocytes) does not differ from normal controls and the abso-
lute number of reticulated platelets is significantly low-
ered.134 Measurement of reticulated platelets has been used
as an aid in assessing bone marrow recovery after bone
marrow transplantation.138 In addition, measurement of re-
ticulated platelets may be useful in evaluating both treat-
ment response and thrombotic risk in patients with throm-
bocytosis.139
However, there are persistent methodological issues
with the flow cytometric measurement of reticulated plate-
lets. Because thiazole orange also binds to ADP and ATP
(contained in dense granules), important controls in the
flow cytometric measurement of reticulated platelets are
the demonstration that thiazole orange staining is a) abol-
ished by pretreatment of the sample with RNAase, and b)
not abolished by pretreatment of the sample with thrombin.
In fact, the higher thiazole orange signal of young platelets
has been reported to be derived to a significant extent from
their large volume and granule content,140-142 leading to the
suggestion that platelet degranulation with TRAP should
be an initial part of the assay for reticulated platelets.140
However, other investigators report that, under modified
assay conditions, degranulation does not significantly
change thiazole orange fluorescence and that RNAase
demonstrates specificity of the thiazole orange staining.136
A new automated method to reliably quantify reticu-
lated platelets, expressed as the immature platelet fraction
(IPF), has been developed using the XE-2100 blood cell
counter with upgraded software (Sysmex, Kobe, Japan).143
The IPF is identified by flow cytometry techniques and the
use of a nucleic acid-specific dye in the reticulocyte/optical
platelet channel. The clinical utility of this parameter was
established in the laboratory diagnosis of thrombocyto-
penia due to increased platelet destruction, e.g. in immune
thrombocytopenic purpura.143
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 Blood Bank Applications patients for whom HLA compatible platelets are not read-
ily available.149.
Quality Control of Platelet Concentrates
Measurement of the platelet surface expression of P-
selectin by flow cytometry is one of the most commonly Platelet-associated IgG
applied measures of platelet activation in platelet concen-
trates stored in blood banks, and efforts have been made to
Measurement of platelet-associated IgG by flow cy-
standardize these measurements.144 However, we14 have
tometry may be useful in immune thrombocytopenias150,151
demonstrated in a non-human primate model that infused,
and alloimmunization.152 A quantitative direct platelet
degranulated platelets rapidly lose surface P-selectin to the
immunofluorescence test for platelet-associated IgG has
plasma pool, but continue to circulate and function in vivo.
been proposed as a screening test for immune thrombocy-
Thus, platelet surface P-selectin molecules, rather than de-
topenias.153
granulated platelets, are rapidly cleared. Our results14 were
subsequently independently confirmed by Berger et al.,25
who found that the platelets of both wild-type and P-
selectin knockout mice had identical life spans. When Platelet Count
platelets were isolated, activated with thrombin, and rein-
jected into mice, the rate of platelet clearance was un-
Platelet Count in Human
changed. The infused thrombin-activated platelets rapidly
The International Council for Standardization in Hae-
lost their surface P-selectin in circulation, and this loss was
matology and the International Society of Laboratory He-
accompanied by the simultaneous appearance of a 100 kDa
matology has recommended a flow cytometric reference
P-selectin fragment in the plasma.25 Storage of platelets at
method for platelet counting that utilizes erythrocyte
4C caused a significant reduction in their life span in vivo,
counts, determined by an automated counter, as an internal
but again no significant differences were observed between
reference standard.154,155
the two genotypes. Thus, the results of Berger et al.25 con-
firm that P-selectin does not mediate platelet clearance.
Furthermore, in a thrombocytopenic rabbit kidney injury
model, Krishnamurti et al.145 reported that thrombin-
activated human platelets: a) lose platelet surface P-selectin Platelet Count in Mice
in the (reticuloendothelial system-inhibited) rabbit circula- With advances in manipulation of the mouse genome,
tion; b) survive in the circulation just as long as fresh hu- murine models have become increasingly important in our
man platelets; and, most importantly, c) are just as effec- understanding of platelet disorders. However, standard
tive as fresh human platelets at decreasing blood loss. methods for murine platelet counting require relatively
In summary, these studies14,25,145 strongly suggest that large volumes of blood and therefore serial platelet counts
the measurement of platelet surface P-selectin in platelet cannot be followed over time in the same mouse. To cir-
concentrates stored in the blood bank should not be used as cumvent this problem, we recently described a rapid, re-
a predictor of platelet survival or function in vivo. How- producible, and accurate flow cytometric method to deter-
ever, platelet surface P-selectin could still be a useful mine the number, and activation state, of circulating plate-
measure of quality control during processing, storage, and lets from a single mouse over extended periods of time.15
manipulation (filtration, washing).144 This is because, in The method uses fluorescent staining of platelets in whole
contrast to the situation in vivo,14,25,145 the activation- blood with a specific antibody and the addition of known
dependent increase in platelet surface P-selectin is not re- numbers of fluorescent beads for standardization of the
versible over time under standard blood banking condi- sample volume. Analysis of platelets obtained by tail
tions.146. bleeding indicated that this sampling procedure did not ac-
tivate platelets, and only 5 L of blood were required for
platelet counting. Thus, the method can be used to follow
the number and the activation state of circulating platelets
Other Blood Bank Applications from individual mice over extended periods of time and is
Flow cytometry can also be used to: identify leukocyte applicable to a wide range of murine models of platelet
contamination of platelet concentrates;104 immunopheno- disorders.15
type platelet HPA-1a147 and other polymorphisms;52 detect
maternal and fetal anti-HPA-1a antibodies;148 and cross-
match platelets, which may be useful for alloimmunized
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 Other Research Applications
Platelet Survival, Tracking, and Function In Vivo
Multi-color whole blood flow cytometry can be used to
track platelets in vivo and determine their survival and
function (Figure 2).14,26,156-160 In 3-color flow cytometry,
the fluorescent labeling typically identifies: 1) platelets in
whole blood (e.g. by a phycoerythrin-conjugated GPIb-,
GPIX-, IIb-, or 3-specific monoclonal antibody), 2) the
infused platelets (e.g. by prelabeling with PKH2, or biotin
followed by the ex vivo addition of streptavidin-RED670),
and 3) an activation-dependent monoclonal antibody (e.g.
FITC-conjugated P-selectin-specific, or activated IIb-, or
3-specific, monoclonal antibody). By these means, the in
vivo function of tracked, infused platelets can be deter-
mined at multiple time points by multiple independent as-
says including, for example: participation in platelet aggre-
gation in response to an in vivo wound, exposure of the fi-
brinogen binding site on IIb3, adherence to Dacron in an
arteriovenous shunt, and generation of procoagulant plate-
let-derived microparticles.14 In a recent study in humans,
Hughes et al.161 used 2-color whole blood flow cytometry
with a FITC-conjugated HLA-A2-specific monoclonal an-
tibody to track and characterize transfused platelets by se-
lectin donor/recipient pairs discrepant for HLA-A2.
Monocyte and Neutrophil Procoagulant Activity With
and Without Surface-Adherent Platelets
Monocytes and neutrophils form heterotypic aggregates
with platelets via engagement of platelet surface P-selectin
with leukocyte surface PSGL-1.22 The resultant intracellu-
lar signaling causes the leukocyte surface expression of tis-
sue factor162 and activation of leukocyte surface Mac-1 (in-
tegrin M2, CD11b/CD18).163,164 The activation-
dependent conformational change in monocyte surface
Mac-1165,166 results in the binding of coagulation factor Xa
and/or fibrinogen to Mac-1.167-169 The platelet surface also
binds coagulation factors via exposed negatively charged
phospholipids such as phosphatidylserine.31 Tissue factor
from the vascular wall and platelets forms a complex with
activated coagulation factor VII, facilitating the activation
of factor X. Tissue factor is a key component of monocyte
surface procoagulant activity.170
We have developed whole blood flow cytometry assays
to measure bound tissue factor, coagulation factor Xa, fi-
brinogen, activated Mac-1 and CD11b on the surface of
monocytes and neutrophils, allowing independent analysis
of monocytes and neutrophils with and without surface-
adherent platelets.171 These methods will be applicable to
future in vivo and in vitro studies of pharmacological
agents targeted to either coagulation and/or cellular activa-
tion processes. The monocyte and neutrophil surface bind-
ing of tissue factor, coagulation factor Xa, and fibrinogen
is mainly dependent on platelet adherence to the mono-
cytes and neutrophils, whereas the monocyte and neutro-
phil surface expression of CD11b and activated Mac-1 is
mainly independent of platelet adherence to the monocytes
and neutrophils.171
Calcium Flux
In washed platelet preparations, platelet-rich plasma, or
whole blood, flow cytometry can be used to measure plate-
let calcium flux, an important platelet second messenger.172
F-Actin
Platelet cytoskeletal rearrangement can be analyzed
flow cytometrically by measuring F-actin content with
NBD- or bodipy-phallicidin or FITC-phalloidin.173,174
Signal Transduction
Flow cytometry can be used to investigate platelet sig-
nal transduction. For example, flow cytometry can be used
to detect and quantify specific intracellular platelet protein
phosphorylation using phosphorylation-specific mono-
clonal antibodies.175
Fluorescence Resonance Energy Transfer
Fluorescence resonance energy transfer (FRET) can be
used to investigate the spatial separation or orientation of
exoplasmic domains within receptor molecules,176 and to
detect and characterize anti-platelet antibodies directed
against HLA class 1 molecules or platelet-specific glyco-
proteins.177
Platelet Recruitment
A three-color flow cytometric method can be used for
the simultaneous monitoring of two platelet populations
that enables the study of the effects of stimuli (even very
short-acting stimuli such as nitric oxide) on platelet re-
cruitment.178
Bacteria-Platelet Interactions
The binding of platelets to other cells, e.g. bacteria, and
the functional consequences of this binding on both cell
types, can be studied by multi-color flow cytometry.179
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Table 1. Applications of flow cytometry to the study of platelets.

Measurement of Platelet Activation (Circulating Blood Bank Applications

Activated Platelets, Quality control of platelet concentrates
Platelet Hyperreactivity, or Platelet Hyporeactivity) Identification of leukocyte contamination in
Activation-dependent monoclonal antibodies platelet concentrates
Leukocyte-platelet aggregates Immunophenotyping of platelet HPA-1a
Platelet-derived microparticles Detection of maternal and fetal anti-HPA-1a
Platelet-platelet aggregates antibodies
Shed blood Platelet cross-matching
Diagnosis of Specific Disorders Platelet-associated IgG
Bernard-Soulier syndrome Immune thrombocytopenias
Glanzmann thrombasthenia Alloimmunization
Storage pool disease Platelet Counting
Heparin-induced thrombocytopenia Other Research Applications
Monitoring of Antiplatelet Agents Platelet survival, tracking, and function in vivo
Thienopyridines Calcium flux
GPIIb-IIIa antagonists F-actin
Aspirin Signal transduction
Monitoring of Thrombopoiesis Fluorescence resonance energy transfer
Reticulated Platelets Platelet recruitment
Bacteria- platelet interactions

Table 2. A typical schema of sample preparation for analysis of platelets by whole blood flow cytometry.

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Table 3. Activation-dependent monoclonal antibodies, i.e. antibodies that bind to activated but not resting platelets.

Activation-dependent platelet surface Prototypic antibodies References

change

Conformational changes in integrins

Activation-induced conformational
17
change in integrin IIb3 resulting in PAC1
exposure of the fibrinogen binding site

Ligand-induced binding sites (LIBS) on

18,108,180
integrin IIb3 PM 1.1, LIBS1, LIBS6

Receptor-induced binding sites (RIBS)

19,34,181
on bound fibrinogen 2G5, 9F9, F26

Activation-induced conformation
change in integrin 21 resulting in
exposure of the collagen binding site IAC-1 182

Exposure of granule membrane proteins

183-185
P-selectin (-granules) S12, AC1.2, 1E3
186,187
GMP-33 (-granules) RUU-SP 1.77
188
CD63 (lysosomes) CLB-gran/12
189
LAMP-1 (lysosomes) H5G11

190
LAMP-2 (lysosomes) H4B4

191
CD40 ligand TRAP1

192
Lectin-like oxidized LDL receptor-1 JTX68
(LOX-1)

Platelet surface binding of secreted

platelet proteins
193,194
P8, TSP-1
Thrombospondin
JS-1 195,196
Multimerin

Development of a procoagulant surface*

29
Factor V/Va binding V237

Factor X/Xa binding 5224 197

30
Factor VIII binding
1B3
*Development of a procoagulant platelet surface can also be detected by the binding of annexin V to phosphatidylserine.31

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