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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 Clinical studies that utilize flow cytometric assays of


Platelets Flow cytometry 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-
Introduction
try by Shattil et al.1 was therefore a major step towards the
Flow cytometry, a remarkably versatile tool for the application of flow cytometry to clinical settings. A typi-
study of platelet function, encompasses multiple assays for cal schema of sample preparation for whole blood flow cy-
multiple purposes (Table 1). tometric analysis of platelets is shown in Table 2. The an-
Flow cytometry rapidly measures the specific charac- ticoagulant is usually buffered sodium citrate, although
teristics of a large number of individual cells. Before flow other anticoagulants can be used.2 The purpose of the ini-
cytometric analysis, single cells in suspension are fluores- tial dilution is to minimize the formation of platelet aggre-
cently labeled, typically with a fluorescently conjugated gates.2 A minimum of 2 monoclonal antibodies is used,
monoclonal antibody. In the flow cytometer, the sus- each conjugated with a different fluorophore. A wide vari-
pended cells pass through a flow chamber and, at a rate of ety of fluorophores are available for antibody conjugation
up to 2000 cells per second, through the focused beam of a (e.g. phycoerythrin, fluorescein, peridinin chlorophyll pro-
laser. After the laser light activates the fluorophore at the tein [PerCP], phycoerythrin-Cy5, phycoerythrin-Texas Red
excitation wavelength, detectors process the emitted fluo- [RED-670], allophycocyanin [APC]). The test mono-
rescence and light scattering properties of each cell. The clonal antibody (recognizing the antigen to be measured) is
intensity of the emitted light is directly proportional to the added at a saturating concentration. The "platelet identi-
antigen density or the characteristics of the cell being fier" monoclonal antibody (e.g. glycoprotein [GP] Ib-,
measured.
GPIX-, integrin IIb-, or integrin 3-specific) is added at a

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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.
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FAX: +1-508-856-4282. e-mail: michelson@platelets.org.


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near saturating concentration. Physiological agonists can Measurement of Platelet Activation
be used in the assay, including thrombin, thrombin recep-
tor-activating peptide (TRAP), adenosine diphosphate
(ADP), collagen, the complement fraction C5b-9, and In the absence of an added exogenous platelet agonist,
thromboxane A2 analogs. Non-physiologic agonists in- whole blood flow cytometry can determine the activation
clude phorbol myristate acetate and calcium ionophore state of circulating platelets, as judged by the binding of an
A23187. Samples are stabilized by fixation, typically with activation-dependent monoclonal antibody. In addition to
a final concentration of 1% paraformaldehyde. The anti- this assessment of platelet function in vivo, inclusion of an
bodies can be added after fixation, provided fixation does exogenous agonist in the assay enables analysis of the reac-
not interfere with antibody binding.2,3 Samples are then tivity of circulating platelets in vitro. In the latter applica-
analyzed in a flow cytometer. After identification of plate- tion, whole blood flow cytometry is a physiological assay
lets both by their characteristic light scatter and by (for ex- of platelet function in that an agonist results in a specific
ample) phycoerythrin positivity, binding of the (for exam- functional response by the platelets: a change in the surface
ple) fluorescein isothiocyanate (FITC)-conjugated test expression of a physiological receptor (or other antigen or
monoclonal antibody is determined by analyzing 5,000 - bound ligand), as determined by a change in the binding of
10,000 individual platelets. Refs. 4,5 contain specific meth- a monoclonal antibody. In addition, as discussed below,
odological protocols of whole blood flow cytometric as- whole blood flow cytometric enumeration of monocyte-
says of platelet function, together with a discussion of platelet aggregates and procoagulant platelet-derived mi-
methodological issues. croparticles are also sensitive markers of in vivo platelet
There are many advantages to flow cytometric analysis of activation.
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- Markers of Platelet Activation
tual in vitro activation and potential loss of platelet sub-
populations.1,8-10 Both the activation state of circulating 1. Activation-Dependent Monoclonal Antibodies
platelets and the reactivity of circulating platelets can be Laboratory markers of platelet activation include acti-
determined. The flow cytometric method permits the de- vation-dependent conformational changes in integrin
tection of a spectrum of specific activation-dependent IIb3 (the GPIIb-IIIa complex, CD41/CD61), exposure
modifications in the platelet surface membrane. Further- of granule membrane proteins, platelet surface binding of
more, as new monoclonal antibodies directed against novel secreted platelet proteins, and development of a procoagu-
functional epitopes are developed, they can easily be in- lant surface (Table 3). The two most widely studied types
corporated into the assay. A subpopulation of as few as of activation-dependent monoclonal antibodies are those
1% partially activated platelets can be detected by whole directed against conformational changes in IIb3 and
blood flow cytometry.10,11 Only minuscule volumes (~5 those directed against granule membrane proteins.
L) of blood are required1,8 making whole blood flow cy-
Integrin IIb3 is a receptor for fibrinogen and von
tometry particularly advantageous for neonatal studies.12
Willebrand factor that is essential for platelet aggrega-
The platelets of patients with profound thrombocytopenia
tion.16 Whereas most monoclonal antibodies directed
can also be accurately analyzed. Finally, flow cytometric
against IIb3 bind to resting platelets, monoclonal anti-
evaluation of platelets is easily adaptable to animal studies
body PAC1 is directed against the fibrinogen binding site
provided the antibody reagents are available.13-15
There are some disadvantages to flow cytometric exposed by a conformational change in IIb3 of activated
analysis of platelet function. First, flow cytometers are ex- platelets (Table 3).17 Thus, PAC1 only binds to activated
pensive instruments to purchase and maintain. Second, for platelets, not to resting platelets. Other IIb3-specific ac-
a clinical assay, sample preparation can be quite compli- tivation-dependent monoclonal antibodies are directed
cated, although the development of kits (e.g. BioCytex, against either ligand-induced conformational changes in
Marseilles, France) has simplified some of the assays. IIb3 (ligand-induced binding sites, LIBS)18 or receptor-
Third, to avoid ex vivo platelet activation, blood samples induced conformational changes in the bound ligand (fi-
should be processed within approximately 30 minutes of brinogen) (receptor-induced binding sites, RIBS)19 (Table
drawing for many assays.1 For the evaluation of some 3). Rather than IIb3-specific monoclonal antibodies,
platelet receptors, this time issue can be circumvented by fluorescein-conjugated fibrinogen can also be used in flow
immediate fixation.2 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 IIb (CD41)-specific monoclonal antibody 5B12 in the neutrophil region (lower
right panel). SSC-H, side scatter height
platelet granules must also be considered in these assays.
The most widely studied type of activation-dependent Tracking of autologous infused biotinylated platelets in
monoclonal antibodies directed against granule membrane baboons by three color whole blood flow cytometry en-
proteins are P-selectin (CD62P)-specific. P-selectin is a abled us26 to directly demonstrate in vivo (Figure 2) that: 1)
component of the granule membrane of resting platelets platelets degranulated by thrombin very rapidly (within 1
that is only expressed on the platelet surface membrane af- minute) form circulating aggregates with monocytes and
ter granule secretion.22 Therefore a P-selectin-specific neutrophils; 2) the percent of monocytes with adherent in-
monoclonal antibody only binds to degranulated platelets, fused platelets is greater than the percent of neutrophils
not to resting platelets. The activation-dependent increase with adherent infused platelets; and 3) the in vivo half-life
in platelet surface P-selectin is not reversible over time in of detectable circulating monocyte-platelet aggregates (ap-
vitro.23,24 However, in vivo circulating degranulated plate- proximately 30 minutes) is longer than both the in vivo
lets rapidly lose their surface P-selectin, but continue to half-life of neutrophil-platelet aggregates (approximately 5
circulate and function.14,25 Platelet surface P-selectin is minutes) and the previously reported14 rapid loss of surface
therefore not an ideal marker for the detection of circulat- P-selectin from non-aggregated infused platelets.
ing degranulated platelets, unless a) the blood sample is All these findings suggested that measurement of circu-
drawn immediately distal to the site of platelet activation, lating monocyte-platelet aggregates may be a more sensi-
b) the blood sample is drawn within 5 minutes of the acti- tive indicator of in vivo platelet activation than either circu-
vating stimulus, or c) there is continuous activation of lating neutrophil-platelet aggregates or circulating P-
platelets. The length of time that other activation- selectin-positive non-aggregated platelets. We therefore
dependent surface markers remain expressed on the platelet performed 2 clinical studies in patients with acute coronary
surface in vivo has not yet been definitively determined. syndromes.26 First, after percutaneous coronary interven-
tion (PCI), there was an increased number of circulating
2. Leukocyte-Platelet Aggregates monocyte-platelet (and, to a lesser extent, neutrophil-
P-selectin mediates the initial adhesion of activated platelet) aggregates, but not P-selectin-positive platelets, in
platelets to monocytes and neutrophils via the P-selectin peripheral blood. Second, of patients presenting to an
glycoprotein ligand 1 (PSGL-1) counter-receptor on the Emergency Department with chest pain, patients with acute
leukocyte surface.22 Monocyte-platelet and neutrophil- myocardial infarction had more circulating monocyte-
platelet aggregates are readily identified by whole blood platelet aggregates than patients without acute myocardial
flow cytometry (Figure 1). 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.

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
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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 Because of the minuscule volumes of blood required,
platelets by some agonists (e.g. C5b-9, collagen/thrombin, whole blood flow cytometry can be used to analyze the
and the calcium ionophore A23187) in the presence of ex- shed blood that emerges from a standardized bleeding time
tracellular calcium ions results in platelet-derived mi- wound.8,34-36 The time-dependent increase in the platelet
croparticles (defined by low forward angle light scatter and surface expression of P-selectin in this shed blood reflects
binding of a platelet-specific monoclonal antibody) that are in vivo activation of platelets.8,34-36 Immediate fixation
procoagulant (determined by binding of monoclonal anti- (prior to antibody incubation) is obligatory, in order to ob-
bodies to activated factors V or VIII or by annexin V).29-31 serve these time-dependent changes. The assay can be
These findings suggest that procoagulant platelet-derived used to demonstrate deficient platelet reactivity in response
microparticles may have an important role in the assembly to an in vivo wound, for example during cardiopulmonary
of the "tenase" and "prothrombinase" components of the bypass.35 In addition, by tracking of infused platelets with
coagulation system in vivo. A flow cytometric method for biotin or PKH2 (see Section IX.A below), shed blood can
the direct detection of procoagulant platelet-derived mi- be used to detect the functional participation of the infused
croparticles in whole blood has been developed.32 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-
Platelet Activation in Clinical Disorders
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 1. Acute Coronary Syndromes
fluorescence per individual particle, irrespective of whether Platelets play an important role in the pathogenesis of
the particle is a single platelet or an aggregate of an un- coronary artery disease, including unstable angina and
known number of platelets. However, a rough estimate of acute myocardial infarction.37 Whole blood flow cytomet-
aggregate size can be made by analyzing the increased ric studies have demonstrated circulating activated plate-
platelet-specific fluorescence. 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 Enhanced systemic platelet degranulation is associated
blood.41,42 with progression of intima-media thickness of the common
Flow cytometric analysis of platelet activation- carotid artery disease in patients with,73 or without,74 type 2
dependent markers can be used to determine optimal anti- diabetes (as determined by platelet surface CD63 and
platelet therapy in clinical settings, e.g. in acute coronary CD40 ligand, and by platelet surface P-selectin, respec-
syndromes43,44 and after coronary stenting.45,46 Flow cy- tively).
tometric analysis of platelet activation markers before PCI
can predict an increased risk of acute and subacute 3. Peripheral Vascular Disease
ischemic events after PCI.47-50 Flow cytometrically de- Circulating activated platelets and platelet hyperreactiv-
tected exposure of LIBS is strongly associated with the de- ity (as determined by P-selectin expression, platelet aggre-
velopment and progression of heart transplant vasculopa- gates, and platelet-derived microparticle formation) are in-
thy.51 creased in patients with peripheral arterial disease com-
The PlA2 polymorphism of GPIIIa has been reported to pared with healthy volunteers.75,76 Circulating monocyte-
be associated with ischemic coronary syndromes.52 Flow and neutrophil-platelet aggregates are significantly greater
cytometry has been used to demonstrate that a) PlA2- in the early postoperative period in patients with peripheral
positive platelets display a lower threshold for activation, vascular disease who go on to develop later graft occlu-
and b) platelets heterozygous for PlA alleles show increased sion.77
sensitivity to antiplatelet drugs.53 With regard to peripheral venous disease, Peyton et
Circulating leukocyte-platelet aggregates are increased al.28 demonstrated an increased presence of monocyte-
in stable coronary artery disease,27,53 unstable angina,38 platelet aggregates in the lower extremity veins of patients
acute myocardial infarction,26,54-56 and cardiopulmonary with chronic venous stasis ulceration, as compared to con-
bypass.57 Circulating leukocyte-platelet aggregates also trol individuals without venous disease. Interestingly,
increase after PCI,26 with a greater magnitude in patients these changes were present not only in blood drawn from
experiencing late clinical events.58 As discussed in Section the lower extremity veins of affected individuals, but also
II.A.2 above, circulating monocyte-platelet aggregates (but in blood drawn from arm veins, suggesting that the
not neutrophil-platelet aggregates) are a more sensitive changes are systemic rather than localized to the lower ex-
marker of in vivo platelet activation than platelet surface P- tremities.28 Powell et al.78 further characterized these find-
selectin in the clinical settings of stable coronary artery ings as being related to the presence of chronic venous dis-
disease,27 PCI,26 and acute myocardial infarction.26 Fur- ease rather than the presence of venous ulceration, since
thermore, circulating monocyte-platelet aggregates are an increased numbers of monocyte-platelet aggregates were
early marker of acute myocardial infarction.56 noted in patients with all classes of venous disease, not just
Platelet-derived microparticles are increased in acute in patients with deep venous valvular insufficiency. Fur-
coronary syndromes59 and cardiopulmonary bypass.34,60 thermore, increased levels of monocyte-platelet aggregates
were noted even in patients with only superficial venous
2. Cerebrovascular Ischemia stasis disease manifested by the presence of varicose veins.
Platelets play an important role in the pathogenesis of Even more intriguing is the fact that the number of mono-
ischemic cerebrovascular disease.61 Increased circulating cyte-platelet aggregates remains elevated six weeks after
P-selectin-positive, CD63-positive, activated IIb3- total correction of the venous insufficiency by stripping of
positive platelets, platelet-derived microparticles, and the abnormal veins, leaving normal venous physiology as
monocyte-platelet aggregates have been reported in acute documented by postoperative duplex scanning.79 This
cerebrovascular ischemia.62-68 This platelet activation is finding suggests an underlying predisposition to the devel-
evident 3 months after the acute event, suggesting the pos- opment of chronic venous disease in these patients, perhaps
sibility of an underlying prothrombotic state.64,66-69 Fur- mediated by monocyte-platelet interactions.
thermore, increased expression of surface P-selectin on
platelets is a risk factor for silent cerebral infarction in pa- 4. Other Clinical Disorders Associated with Platelet Hyper-
tients with atrial fibrillation.70 reactivity and/or Circulating Activated Platelets
Platelet-derived microparticles are increased after tran- There are numerous other conditions in which whole
sient ischemic attacks.34,71 Increased platelet-derived mi- blood flow cytometric measurement of platelet hyperreac-
croparticles and procoagulant activity occur in sympto- tivity, circulating activated platelets, and/or circulating
matic patients with prosthetic heart valves and provided a leukocyte-platelet aggregates may prove to have a clinical
potential pathophysiological explanation of cerebrovascu- role, including diabetes mellitus,73,80-82 cystic fibrosis,83
lar events in this patient group.72 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- Diagnosis of Specific Disorders
ple organ dysfunction syndrome,92,93 antiphospholipid syn-
drome,94 systemic lupus erythematosus,94 rheumatoid ar-
thritis,94,95 inflammatory bowel disease,96 myeloprolifera- Platelet Surface Glycoprotein Deficiencies
tive disorders,97,98 and Alzheimer disease.99 High levels of
circulating monocyte-platelet aggregates can predict rejec- 1. Bernard-Soulier Syndrome
tion episodes after orthotopic liver transplantation.100 Bernard-Soulier syndrome is an inherited deficiency of
Uremic patients with thrombotic events have higher num- the GPIb-IX-V complex.105 Flow cytometric analysis with
bers of circulating platelet-derived microparticles than GPIb-, GPIX-, and GPV-specific monoclonal antibodies
those without thrombotic events.101 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
Reduced Circulating Activated Platelets and Platelet syndrome platelets from similarly-sized red and white
Hyporeactivity blood cells. Because light scatter (especially forward light
In addition to the detection of increased circulating ac- scatter) correlates with platelet size, light scatter gates may
tivated platelets and platelet hyperreactivity discussed in need to be adjusted in the flow cytometric analysis of giant
Sections II.B above, whole blood flow cytometry may be platelet syndromes such as Bernard-Soulier syndrome.
useful in the clinical assessment of reduced circulating ac- This adjustment may result in overlap of the light scatter of
tivated platelets and platelet hyporeactivity although giant platelets with red and white blood cells. It is there-
there are few published studies in this area. fore essential to include in the assay a platelet-specific
monoclonal antibody as a platelet identifier. For Bernard-
1. Very Low Birth Weight Preterm Neonates Soulier syndrome platelets, this identifier antibody obvi-
Compared to adults, the platelets of very low birth ously cannot be GPIb-, GPIX-, or GPV-specific.
weight preterm neonates are markedly hyporeactive to
thrombin, ADP/epinephrine and thromboxane A2, as de- 2. Glanzmann Thrombasthenia
termined by flow cytometric detection of a) the exposure of Glanzmann thrombasthenia is an inherited deficiency
the fibrinogen binding site on IIb3, b) fibrinogen bind- of integrin IIb3.105 Flow cytometric analysis with IIb-
ing, c) the increase in platelet surface P-selectin, and d) the
and 3-specific monoclonal antibodies provides a rapid
decrease in platelet surface GPIb.12 Furthermore, proco-
and simple means for the diagnosis of the homozygous and
agulant platelet-derived microparticles are decreased in
heterozygous states of Glanzmann thrombasthenia.106,107
preterm neonates compared with adults.32 This platelet hy-
In addition, a panel of activation-dependent monoclonal
poreactivity of preterm neonates, as judged by both activa-
antibodies can be used to evaluate patients with defects in
tion-dependent platelet surface changes and the generation
platelet aggregation,108 secretion,109 or procoagulant activ-
of procoagulant platelet-derived microparticles, may con-
ity.110
tribute to the propensity of very low birth weight neonates
to intraventricular hemorrhage.102

2. Acute Myeloid Leukemia


Prediction of hemorrhage in thrombocytopenic patients Storage Pool Disease
has been problematic because of the lack of laboratory Inherited dense granule storage pool deficiency, a rela-
markers or validated clinical assessment tools.103 The tively common cause of a mild hemorrhagic diathesis, can-
clinical decision to prophylactically transfuse platelets is not be reliably diagnosed by standard platelet aggregome-
therefore usually based solely on the platelet count.104 try.111 The conventional method to establish the diagnosis
However, low levels of expression of platelet surface P- of storage pool disease is to label the platelets with the
selectin, as determined by whole blood flow cytometry, fluorescent dye mepacrine and then measure platelet fluo-
have recently been reported to be a prognostic marker for rescence by microscopy.112 This assay is based on the se-
hemorrhage in acute myeloid leukemia.103 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 Monitoring of Thrombopoiesis
fluorescent light microscopic methods, but improves the
detection of the mepacrine-loaded platelets by quantita-
tively measuring fluorescence on a large number (5000) of Whole blood flow cytometric methods have been de-
platelets113,114 Acquired dense granule storage pool defi- veloped for the identification of young platelets (i.e. those
ciency, which occurs in myeloproliferative disorders and containing mRNA) by their staining with thiazole or-
end-stage renal failure, can also be diagnosed by flow cy- ange.134-136 Because of the analogy to reticulocytes, these
tometry.113,115 An alternative approach to the flow cy- thiazole orange-positive platelets have been termed "reticu-
tometric diagnosis of storage pool disease is to measure in- lated platelets"137 and have been used to monitor throm-
traplatelet serotonin (5-hydroxytryptamine) with a sero- bopoiesis.135 Thrombocytopenic patients whose bone mar-
tonin-specific monoclonal antibody.116 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-
Heparin-Induced Thrombocytopenia yocytes) does not differ from normal controls and the abso-
Unlike normal sera and sera from patients with quinine- lute number of reticulated platelets is significantly low-
or quinidine-induced thrombocytopenia, sera from patients ered.134 Measurement of reticulated platelets has been used
with heparin-induced thrombocytopenia (HIT) generate as an aid in assessing bone marrow recovery after bone
procoagulant platelet-derived microparticles from normal marrow transplantation.138 In addition, measurement of re-
platelets.117 This observation has been used to develop a ticulated platelets may be useful in evaluating both treat-
rapid, specific, and sensitive flow cytometric assay for the ment response and thrombotic risk in patients with throm-
diagnosis of HIT.118 Alternative flow cytometric ap- bocytosis.139
proaches to the diagnosis of HIT have been described.119 However, there are persistent methodological issues
with the flow cytometric measurement of reticulated plate-
lets. Because thiazole orange also binds to ADP and ATP
Monitoring of Antiplatelet Agents
(contained in dense granules), important controls in the
flow cytometric measurement of reticulated platelets are
the demonstration that thiazole orange staining is a) abol-
The in vivo effect of the thienopyridines (clopidogrel ished by pretreatment of the sample with RNAase, and b)
and ticlopidine) on platelet function can be monitored by not abolished by pretreatment of the sample with thrombin.
the vasodilator-stimulated phosphoprotein (VASP) assay In fact, the higher thiazole orange signal of young platelets
(BioCytex, Marseilles, France).120,121 Through the use of has been reported to be derived to a significant extent from
arachidonic acid as the agonist, flow cytometry can also be their large volume and granule content,140-142 leading to the
used to monitor aspirin therapy.122,123 suggestion that platelet degranulation with TRAP should
Flow cytometric methods can be used to monitor be an initial part of the assay for reticulated platelets.140
GPIIb-IIIa antagonists (abciximab, eptifibatide and tirofi- However, other investigators report that, under modified
ban) by measuring receptor occupancy by these drugs. assay conditions, degranulation does not significantly
These methods can be categorized as either direct or indi- change thiazole orange fluorescence and that RNAase
rect. One type of direct method is a competitive binding demonstrates specificity of the thiazole orange staining.136
assay with either: a) biotinylated124 or FITC-conjugated125 A new automated method to reliably quantify reticu-
GPIIb-IIIa antagonists; b) blocking monoclonal antibod- lated platelets, expressed as the immature platelet fraction
ies;126,127 c) peptides with very high affinities for GPIIb- (IPF), has been developed using the XE-2100 blood cell
IIIa (disintegrins128 or cyclic RGD peptides129); or, d) fi- counter with upgraded software (Sysmex, Kobe, Japan).143
brinogen binding after platelet activation (detected by a The IPF is identified by flow cytometry techniques and the
polyclonal anti-fibrinogen antibody).130 Another type of use of a nucleic acid-specific dye in the reticulocyte/optical
direct method measures the binding of an antibody directed platelet channel. The clinical utility of this parameter was
against the GPIIb-IIIa antagonist.131,132 Indirect methods established in the laboratory diagnosis of thrombocyto-
measure either: a) the GPIIb-IIIa antagonist-induced bind- penia due to increased platelet destruction, e.g. in immune
ing of an anti-LIBS antibody;133 or, b) platelet aggregation, thrombocytopenic purpura.143
as determined by light scatter.124
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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 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-
Platelet Survival, Tracking, and Function In Vivo phil surface expression of CD11b and activated Mac-1 is
Multi-color whole blood flow cytometry can be used to mainly independent of platelet adherence to the monocytes
track platelets in vivo and determine their survival and and neutrophils.171
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 Calcium Flux
infused platelets (e.g. by prelabeling with PKH2, or biotin In washed platelet preparations, platelet-rich plasma, or
followed by the ex vivo addition of streptavidin-RED670), whole blood, flow cytometry can be used to measure plate-
and 3) an activation-dependent monoclonal antibody (e.g. let calcium flux, an important platelet second messenger.172
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- F-Actin
mined at multiple time points by multiple independent as- Platelet cytoskeletal rearrangement can be analyzed
says including, for example: participation in platelet aggre- flow cytometrically by measuring F-actin content with
gation in response to an in vivo wound, exposure of the fi- NBD- or bodipy-phallicidin or FITC-phalloidin.173,174
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, Signal Transduction
Hughes et al.161 used 2-color whole blood flow cytometry Flow cytometry can be used to investigate platelet sig-
with a FITC-conjugated HLA-A2-specific monoclonal an- nal transduction. For example, flow cytometry can be used
tibody to track and characterize transfused platelets by se- to detect and quantify specific intracellular platelet protein
lectin donor/recipient pairs discrepant for HLA-A2. phosphorylation using phosphorylation-specific mono-
clonal antibodies.175

Monocyte and Neutrophil Procoagulant Activity With


and Without Surface-Adherent Platelets Fluorescence Resonance Energy Transfer
Monocytes and neutrophils form heterotypic aggregates Fluorescence resonance energy transfer (FRET) can be
with platelets via engagement of platelet surface P-selectin used to investigate the spatial separation or orientation of
with leukocyte surface PSGL-1.22 The resultant intracellu- exoplasmic domains within receptor molecules,176 and to
lar signaling causes the leukocyte surface expression of tis- detect and characterize anti-platelet antibodies directed
sue factor162 and activation of leukocyte surface Mac-1 (in- against HLA class 1 molecules or platelet-specific glyco-
tegrin M2, CD11b/CD18).163,164 The activation- proteins.177
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 Platelet Recruitment
binds coagulation factors via exposed negatively charged A three-color flow cytometric method can be used for
phospholipids such as phosphatidylserine.31 Tissue factor the simultaneous monitoring of two platelet populations
from the vascular wall and platelets forms a complex with that enables the study of the effects of stimuli (even very
activated coagulation factor VII, facilitating the activation short-acting stimuli such as nitric oxide) on platelet re-
of factor X. Tissue factor is a key component of monocyte cruitment.178
surface procoagulant activity.170
We have developed whole blood flow cytometry assays
to measure bound tissue factor, coagulation factor Xa, fi-
Bacteria-Platelet Interactions
brinogen, activated Mac-1 and CD11b on the surface of
The binding of platelets to other cells, e.g. bacteria, and
monocytes and neutrophils, allowing independent analysis
the functional consequences of this binding on both cell
of monocytes and neutrophils with and without surface-
types, can be studied by multi-color flow cytometry.179
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-
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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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