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Light Transmission Aggregometry and ATP Release for the Diagnostic

Assessment of Platelet Function

Article  in  Seminars in Thrombosis and Hemostasis · April 2009

DOI: 10.1055/s-0029-1220324 · Source: PubMed

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 Light Transmission Aggregometry and ATP
Release for the Diagnostic Assessment of
Platelet Function
Marco Cattaneo, M.D.1

ABSTRACT

Light transmission aggregometry (LTA) is the gold standard for the study of
patients with defects of platelet function. Use of LTA in clinical practice for predicting the
risk of thrombosis or monitoring the pharmacologic effects of antiplatelet agents should be
discouraged, because not only is the monitoring of treatment with antiplatelet agents (with
any laboratory test) not indicated at present, but also the lack of standardization of the
technique for LTA makes it additionally unsuitable for this purpose. The need for
standardization of LTA has recently been emphasized by the results of four surveys,
which showed that there is a wide variation in the methodology used worldwide. A
modification of the traditional LTA is the lumiaggregometer, which measures platelet
secretion in parallel with platelet aggregation. This technique is probably preferable to
traditional LTA in the diagnostic workup of patients with inherited defects of platelet
function, because it is more sensitive to the most common disorders, which are charac-
terized by abnormalities of platelet secretion. LTA (or lumiaggregometry) is useful as a first
screening test of patients with the clinical suspicion of defects of platelet function because it
helps to provide an interim diagnostic hypothesis, which can then be confirmed or
discounted using appropriate and specific tests.

KEYWORDS: Platelet function disorder, platelet aggregation, platelet secretion, light

transmission aggregometry, lumiaggregometry

W hen a blood vessel is injured, platelets adhere
to the exposed subendothelium (platelet adhesion), are
activated (platelet activation), and secrete the contents
of their granules (platelet secretion), including the pla-
telet agonists adenosine diphosphate (ADP) and sero-
tonin, which interact with specific platelet receptors to
enhance recruitment of additional platelets to form
aggregates (platelet aggregation). In addition, platelets
play a role in the coagulation mechanism, providing
the necessary surface of procoagulant phospholipids
(platelet procoagulant activity) for assembly of coagula-
tion complexes. Inherited or acquired abnormalities of
platelet number or function are associated with a
heightened risk for bleeding, proving that platelets
play an important role in hemostasis. Typically, pa-
tients with platelet disorders have mucocutaneous
bleeding of variable severity and excessive hemorrhage
after surgery or trauma.1

1
Unità di Medicina III, Ospedale San Paolo, Università di Milano,
Milano, Italy.
Address for correspondence and reprint requests: Prof. Marco
Cattaneo, Unità di Medicina III, Ospedale San Paolo, Università di
Milano, Via di Rudinı̀ 8, 20142 Milano, Italy (e-mail: marco.
cattaneo@unimi.it).
Diagnostic Evaluation of Platelet Function Disorders; Guest
158
Editors, Catherine P.M. Hayward, M.D., Ph.D., F.R.C.P.(C.), and
Emmanuel J. Favaloro, Ph.D., M.A.I.M.S.
Semin Thromb Hemost 2009;35:158–167. Copyright # 2009
by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New
York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI 10.1055/s-0029-1220324. ISSN 0094-6176.

Several kinds of different platelet function tests
have been developed over the years to identify or
diagnose platelet function disorders.2,3 Of the available
methods, light transmission aggregometry (LTA),
which is probably the oldest one, is still considered the
gold standard for the study of patients with presumptive
platelet function disorders.
LIGHT TRANSMISSION AGGREGOMETRY
LTA was developed in 1962 by Born and O’Brien
independently.4,5 LTA measures light transmission
through a sample of platelets in suspension (platelet-
rich plasma [PRP], washed platelets, or gel-filtered
platelets) that increases when platelets are aggregated
by an agonist. LTA is a time-consuming and technically
challenging technique that is affected by many preana-
lytical and analytical variables, and these must be care-
fully controlled for by expert personnel. For this reason,
LTA should be performed only in highly specialized
laboratories.
LTA: Technical Issues
A light transmission aggregometer is a photometer,
consisting of a light source, a cuvette holder incorporat-
ing a rotating magnet (which drives a small stirrer placed
in the platelet suspension), a thermostat heater (which
maintains the temperature of the sample constant), and a
photoelectric cell that receives the light beam after its
passage through the sample of platelet suspension. The
light signal is then transferred electronically to a chart
recorder or a computer.
Maximal optical density occurs when platelets are
in the resting state, evenly distributed throughout the
plasma or the buffered solution. Upon addition of a
platelet agonist to the sample, platelets are activated and
change their shape from discoid to spiny spheres, an
event that is associated with a transient increase in
optical density. The only exception to this rule is
represented by platelet activation by epinephrine, which
does not result in platelet shape change. The transient
increase in optical density of the platelet suspension is
rapidly followed by a brisk increase in light transmission,
which is indicative of ongoing platelet aggregation, the
formation of platelet clumps of different size allowing
the passage of more light through the sample.
The aggregation curve either reaches a plateau of
light transmission, without deflecting toward baseline
(irreversible aggregation), or else tends to return toward
the baseline (reversible aggregation). Occasionally, when
platelet aggregation is stimulated by weak agonists at
critical concentrations, the aggregation curve has a
biphasic appearance: an initial wave of aggregation
(primary wave), followed by a secondary wave of aggre-
gation, which is usually irreversible. Alternatively, the
LIGHT TRANSMISSION AGGREGOMETRY/CATTANEO 159
primary wave may deaggregate if secondary aggregation
does not occur. At higher concentrations of the weak
agonists, the two waves of aggregation fuse, giving rise to
a single, irreversible curve. Epinephrine-induced platelet
aggregation represents an exception to this rule, as, with
normal platelets, the two waves of platelet aggregation,
when present, never fuse into one single wave, even
when epinephrine is used at very high concentrations.
Calibration of the instrument for recording ag-
gregation involves establishing a baseline on the recorder
at between 0 and 10% of pen excursion, using the
unstimulated platelet suspension as the high optical
density standard. Some instruments set this to zero but
will record below this value; otherwise, the choice of 10%
pen excursion will allow the recording of the slight
increase in optical density that ensues after platelet
simulation with an agonist, reflecting platelet shape
change. Maximal light transmission is usually set at a
nominal value of 90 to 100% maximal pen excursion,
using platelet-poor plasma (PPP) or buffered solution as
the high light transmission standard, depending on
whether PRP or platelet suspension in buffered solution
is being tested.
Lumiaggregometer
A modification of the traditional light transmission
aggregometer is the lumiaggregometer, which can
measure platelet secretion in parallel with platelet
aggregation. The method is based on the biolumines-
cent determination of ATP that is released from pla-
telet d-granules, which reacts with the firefly extracts
luciferin and luciferase; the reaction is followed by
oxidative decarboxylation of luciferin, resulting in light
emission. For measurement of aggregation, the lumi-
aggregometer employs a diode that emits light (LED)
in the infrared range, which is detected by a photo-
transistor, whereas, for measurement of luminescence
resulting from ATP secretion, it uses a photomultiplier
tube located at right angles to the light path of the
LED. Measurement of platelet secretion using the
luciferin-luciferase reaction can also be evaluated with
whole blood aggregometry, as explained elsewhere.3
Platelet Agonists
Several agonists are used to stimulate platelet suspen-
sions in a light transmission aggregometer. The most
commonly used agonists include ADP, epinephrine,
collagen, arachidonic acid, and the thromboxane A2
analogue U46619. Another commonly used reagent is
ristocetin, which should not be considered an agonist,
as it induces platelet agglutination, a passive process
during which platelets form clumps without being
activated (this accounts for the lack of platelet shape
change preceding the increase in light transmission
160 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 35, NUMBER 2 2009

induced by ristocetin). When ristocetin-induced agglu-
tination of normal platelets in citrated PRP reaches a
critical level, platelets synthesize thromboxane A2
(TxA2), which induces platelet secretion and platelet
aggregation; it must be noted, however, that TxA2
synthesis is not triggered by ristocetin directly, but by
the close platelet-to-platelet contact that is brought
about by platelet agglutination (see later). Ristocetin is
useful to diagnose abnormalities of the interaction
between the platelet glycoprotein Ib (GPIb) and von
Willebrand factor (VWF), and thus critical for evalua-
tion of Bernard-Soulier syndrome (BSS) and some
forms of von Willebrand disease (VWD). Many other
agonists are available for studies of platelet function in
the clinical setting and, more commonly, for research
purposes (e.g., thrombin, the thrombin receptor [PAR-1]
activating peptide [TRAP], convulxin, the calcium ion-
ophore A23187, g-thrombin, platelet activating factor
[PAF]-acether, vasopressin, and others).
The aggregation response of platelets to an ago-
nist is amplified by the production of TxA2 from
membrane phospholipids and by the secretion of ADP
from the platelet d-granules: both TxA2 and ADP are
aggregating agents, which, by interacting with their
specific receptors, amplify the aggregation response of
platelets (Fig. 1). Therefore, the maximal extent of
platelet aggregation that is observed in a light trans-
mission aggregometer after platelet stimulation with an
agonist is the sum of the platelet aggregation induced
directly by the agonist, plus the contributions brought
about by endogenous TxA2 and released ADP.
There are two types of platelet agonists: strong
agonists and weak agonists.6 Strong agonists (e.g., colla-
gen, thrombin, TxA2) directly induce platelet aggrega-
tion, TxA2 synthesis, and platelet granule secretion,
whereas weak agonists (e.g., ADP, epinephrine) directly
induce platelet aggregation without triggering secretion
(Fig. 1). Strong agonists, when used at sufficiently low
concentrations, may behave like weak agonists, whereas
weak agonists never behave like strong agonists, even
when they are used at extremely high concentrations
(Fig. 2). However, platelet secretion can sometimes
follow aggregation induced by a weak agonist, when
the synthesis of endogenous TxA2 is triggered by the
close platelet-to-platelet contact that occurs during pla-
telet aggregation. This event (which occurs also when
the platelet-to-platelet contact is induced by ristocetin or
other agglutinating agents) is artifactually enhanced by
reducing the levels of ionized calcium in the medium
([Ca2þ]o) to micromolar levels, like in citrated PRP.7,8
As a matter of fact, the event can be observed in most
normal citrated PRP samples, but it does not occur in the
vast majority of normal PRP samples collected in anti-
coagulants that do not decrease the physiologic [Ca2þ]o,
such as direct thrombin inhibitors.9 The best demon-
stration that platelet secretion is not induced by the weak

Figure 1 The aggregation response of platelets to agonists is amplified by the production of TxA2 from membrane
phospholipids and the secretion of ADP from platelet d-granules: both TxA2 and ADP are aggregating agents, which, by
interacting with their specific receptors, amplify the aggregation response of platelets. In addition, secreted ADP, by interacting
with its receptor P2Y12, amplifies platelet secretion induced by strong agonists.29 There are two types of platelet agonists:
strong agonists and weak agonists. Strong agonists directly induce platelet aggregation, TxA2 production, and platelet
secretion. In contrast, weak agonists directly induce platelet aggregation only: when platelet aggregation reaches a threshold
level, it triggers TxA2, which causes platelet secretion and, in collaboration with secreted ADP, secondary aggregation. The
aggregation-dependent TxA2 production is artifactually enhanced in citrated PRP, where the concentration of ionized calcium in
the medium ([Ca2þ]o) is decreased to micromolar levels.
 LIGHT TRANSMISSION AGGREGOMETRY/CATTANEO 161

Figure 2 Responses of normal human citrated PRP to strong agonists, weak agonists, and agglutinating agents measured in
a lumiaggregometer: upper tracings reflect platelet secretion of ATP (luminescence; roman fonts); lower tracings reflect platelet
aggregation (light transmission; italic fonts). Left tracings (strong agonist): The TxA2 analogue U46619 (1 mmol/L), a strong
agonist, directly induces platelet secretion, as demonstrated by the observations that platelet secretion occurred
(i) simultaneously with platelet aggregation (b), (ii) under experimental conditions that prevented the formation of platelet
aggregates (no stirring of the PRP sample in the lumiaggregometer) (c). At low concentration (0.1 mmol/L), U46619 behaved like
a weak agonist (see later), as platelet secretion occurred 1.5 minutes after stimulation, when platelet aggregation reached a
threshold level (a) and did not occur in the absence of platelet aggregation (no stirring of PRP sample in the lumiaggregometer)
(not shown in this figure). All platelet responses to U46619 (including platelet shape change, which is evidenced by the slight
decrease in light transmission and the loss of oscillations in baseline tracings immediately after stimulation of PRP) were
inhibited by prostaglandin E1 (PGE1) (d), which abolishes platelet reactivity to agonists by increasing the cytoplasmic levels of
cyclic AMP. Middle tracings (weak agonist): ADP (a weak agonist) directly induces platelet aggregation only. At low
concentration (2 mmol/L; a), ADP induced biphasic and irreversible wave of platelet aggregation; at higher concentration
(4 mmol/L; b), ADP induced monophasic, irreversible wave of aggregation (the primary and secondary waves fused in one single
wave of aggregation). Despite the differences in platelet aggregation observed at the two concentrations of ADP, no
differences were observed in platelet secretion, which occurred 1.0 minute after the addition of ADP, when platelet
aggregation reached a threshold level: this observation suggests that platelet secretion is not triggered by ADP directly, but,
rather, by platelet aggregation. This is also demonstrated by the observation that ADP, even at very high concentration
(10 mmol/L; c), did not cause platelet secretion when the PRP sample was not stirred in the aggregometer to prevent the
formation of platelet aggregates. All platelet responses to ADP (including platelet shape change) were inhibited by PGE1 (d).
Right tracings (agglutinating agent): Ristocetin (an agglutinating agent) caused an increase of light transmission through the PRP
sample, not preceded by platelet shape change (which is otherwise indicative of platelet activation). A secondary wave of
aggregation, which was paralleled by platelet secretion, occurred 1.0 minute after the addition of low concentration of
ristocetin (0.8 mg/mL; a); the secondary wave of platelet aggregation fused with the primary wave of platelet agglutination
when ristocetin was used at high concentration (1.2 mg/mL; b). No agglutination or secretion was observed when ristocetin (1.2
mg/mL; c) was added to PRP in the absence of stirring. PGE1 (1 mmol/L) abolished the secondary wave of aggregation and
platelet secretion but did not inhibit the primary wave of platelet agglutination induced by ristocetin, at both 0.8 mg/mL (d) and
1.2 mg/mL (not shown). Secondary waves of aggregation and platelet secretion after exposure of platelets to weak agonists or
to agglutinating agents do not occur when the concentration of plasma ionized Ca2þ is physiologic (e.g., when direct thrombin
inhibitors are used as anticoagulants instead of trisodium citrate) (not shown).

agonist (or by agglutinating agents) directly, but reflects
signaling induced by close platelet-to-platelet contact,
comes from the observation that no platelet secretion
occurs from normal human platelets that are stimulated
by ADP or epinephrine (or are exposed to ristocetin),
even at high concentration, under conditions in which
platelet aggregation does not occur: for instance,
when the receptor function for adhesive proteins on
aIIbb3 is inhibited by monoclonal antibodies or
RGD-containing peptides, or, more simply, when the
platelet suspension in the aggregometer is not stirred
(Fig. 2). This contrasts with the observation that strong
agonists induce platelet secretion even when platelet
aggregation is prevented (Fig. 2). The artifactual effect
of low [Ca2þ]o on weak agonist–induced platelet TxA2
production and platelet secretion is demonstrated by the
162 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 35, NUMBER 2
observation that weak agonists elicit minimal or no TxA2
production, platelet secretion, and secondary waves of
aggregation in normal platelets that are suspended in
media containing [Ca2þ]o in the physiologic range.8,9
Albeit artifactual, the TxA2-dependent potentiation of
platelet function induced by weak agonists at low
[Ca2þ]o may be useful for increasing the sensitivity of
LTA to mild defects of platelet function, especially
those that are characterized by abnormalities of platelet
secretion.
Factors Affecting the Results of LTA
Platelets are very reactive cells and are prone to activation
and desensitization during the preparation of the sample
of platelet suspension to be tested. Because both can
significantly alter platelet responsiveness to aggregating
agents, very careful handling of the blood samples is
necessary to minimize the risk of platelet activation and
desensitization.
There are many preanalytical and analytical var-
iables that affect the results of LTA. Some of the most
relevant ones are summarized in the following para-
graphs.
BLOOD SAMPLING
Blood should be collected from a clean, problem-free
venipuncture, using a high-caliber needle (at least
21-gauge), with minimal or no venostasis. The first
few milliliters of blood drawn should be discarded or
used for other tests. Blood should be gently mixed with
the anticoagulant as soon as possible.
ANTICOAGULANTS
In the clinical setting, trisodium citrate (either 109 or
129 mM) is commonly used in the ratio of 1 part to
9 parts of whole blood. Anticoagulants that maintain the
concentration of divalent cations (e.g., Ca2þ, Mg2þ) in
the physiologic range (such as the direct thrombin
inhibitors, hirudin and Phe-Pro-Arg-chloromethylke-
tone [PPACK]) are much more expensive than citrate.
In addition, because the secondary wave of aggregation
induced by weak agonists does not occur with [Ca2þ]o in
the physiologic range, these alternative anticoagulants
would not allow the detection of abnormal responses to
weak agonists, which are typical of some common
platelet function disorders.
PLATELET COUNT
Early studies showed that diluting PRP with autologous
PPP decreases the platelet responses to agonists, thus
suggesting that the platelet count in PRP is one of the
major determinants of platelet responses to agonists.10
As a consequence, it is still common practice to adjust
the platelet count in PRP with autologous PPP to a
standardized value (which usually varies between
2009
200  109/L and 300  109/L) or to match that of a
control sample that should be run in parallel with that of
the patient under study. However, this practice has been
recently criticized. One study showed that the decrease
in platelet aggregation that is observed after dilution of
PRP with PPP is not due to the induced decrease in
platelet count but to the inhibitory effects on platelet
aggregation of substances contained in PPP.11 Other
recent studies suggested that adjustment of platelet
count in PRP is a time-consuming and unnecessary
procedure.12,13
pH
During storage, the pH of PRP samples tends to increase
to levels that may significantly affect the platelet aggre-
gation response14; this effect is mediated by the diffusion
of CO2 out of the sample. Procedures that help mini-
mize this phenomenon include use of a buffered anti-
coagulant, use of tubes of small caliber (to minimize the
surface area exposed to the atmosphere), and keeping
sample tubes capped until tested.
TEMPERATURE
PRP samples should be kept at room temperature before
testing.14,15 However, platelet aggregation tests should
be performed at 378C, as platelet aggregation is sub-
optimal at temperatures below 378C. Therefore, the
cuvette containing the PRP sample should be placed in
a cuvette holder at 378C for at least 3 to 4 minutes before
being tested to reach the desired temperature prior to
testing aggregation.
AGGREGOMETER STIR SPEED
The sample of platelet suspension should be continu-
ously stirred at a constant speed during LTA testing.
Sample stirring helps maintain the platelets in an even
suspension and, most importantly, favors platelet-to-
platelet contact, which is necessary for platelet aggrega-
tion to proceed. Although a stirring speed of 1000 rpm is
usually recommended, stirring speeds between 700 and
1100 rpm are generally acceptable.14 At lower rotating
speeds, the slopes of the aggregation curves tend to
decrease,10 and at higher rotating speeds, platelets may
be traumatized sufficiently to undergo secretion and
spontaneous aggregation.16
TIME
Platelet responses to agonists change with time. Soon
after preparation of PRP, platelets are hyporesponsive,
presumably as a consequence of desensitization of the
ADP receptors, which are exposed to ADP that is
released by platelets and red blood cells during centrifu-
gation of whole blood sample to prepare PRP. Platelet
responsiveness is restored after 15 minutes, increases
further in the following 30 minutes, and remains stable
until about the third hour after PRP preparation.14

Finally, the responsiveness declines again, most likely as
a consequence of exhaustion of energy reserves. Accord-
ingly, platelet function testing by agonist challenge
generally begins some 15 to 30 minutes after the prep-
aration of PRP and should be completed within 3 to
4 hours.
Need for Standardization of LTA
Early attempts to standardize the preanalytical and
analytical variables of LTA17 were unsuccessful, as
shown by the results of several surveys regarding several
details in the methodology, which were distributed to
members of the North American Specialized Coagu-
lation Laboratory Association (NASCOLA),18 to par-
ticipants of the Haematology Quality Assurance
Program of the Royal College of Pathologists of
Australia,19 and to participants of the UK National
External Quality Assessment Scheme (UK NEQAS).20
All surveys showed that there is a wide variation in the
methodology used to perform LTA. These findings
were confirmed by a fourth, larger survey, organized by
the Platelet Physiology subcommittee of the Interna-
tional Society on Thrombosis and Haemostasis Scientific
and Standardization Committee (ISTH SSC) (manu-
script in preparation). In consideration of these shortfalls,
Zhou and Schmaier described one laboratory’s methods
and interpretations of platelet aggregation and secretion
studies of PRP using each of the common platelet
agonists, with the intent to stimulate interest in the
development of professional guidelines for platelet func-
tion testing in the clinical laboratory.21 In addition, the
Clinical and Laboratory Standards Institute (CLSI)
recently issued an approved guideline,22 and the Platelet
Physiology subcommittee of the ISTH SSC is preparing
official guidelines, which will be issued in 2009. Stand-
ardization of platelet function testing and external
quality control for platelet function testing are discussed
in greater detail in other articles within this issue of
Seminars in Thrombosis and Hemostasis.23,24
Clinical Application of LTA
In the clinical setting, LTA should be used for diag-
nosing bleeding disorders that are associated with
inherited or acquired platelet dysfunction. Use of
LTA in clinical practice for predicting the risk of
thrombosis or monitoring the pharmacologic effects
of antiplatelet agents, such as aspirin or clopidogrel,
should be discouraged. Not only is the monitoring of
treatment with antiplatelet agents (with any kind of
laboratory test) not indicated at present,25 but also the
lack of standardization of the technique for LTA makes
it additionally unsuitable for this purpose. In fact, the
results obtained within one laboratory could hardly be
compared with those obtained in a different laboratory,
LIGHT TRANSMISSION AGGREGOMETRY/CATTANEO 163
rendering pointless any attempt to define universal
cutoff values to identify different degrees of response
to antiplatelet therapy or to predict the risk of throm-
bosis. In addition, the intrinsic variability of the tech-
nique remains unsatisfactorily high, even when the
preanalytical and the analytical variables are strictly
controlled for in a single laboratory.
USE OF LTA IN THE DIAGNOSTIC WORKUP
OF INHERITED PLATELET FUNCTION
DISORDERS
Inherited disorders of platelet function have generally
been classified based on their platelet function profiles
or according to the responses that are abnormal.
However, because distinct platelet function processes
are intimately related, a clear distinction between
disorders of platelet adhesion, aggregation, activation,
secretion, and procoagulant activity is, in many instan-
ces, problematic. For example, platelets that are defi-
cient in the glycoprotein (GP) complex Ib/IX/V do not
adhere normally to the subendothelium and for this
reason are generally included in the group of abnor-
malities of platelet adhesion. However, they also do
not undergo normal activation and aggregation at
high shear, do not aggregate normally to thrombin,
and display abnormal procoagulant responses. For this
reason, a classification that is based on abnormalities of
platelet components sharing common characteristics is
probably preferable. Based on this criterion, six groups
of inherited disorders of platelet function can be
identified: (1) abnormalities of platelet receptors for
adhesive proteins; (2) abnormalities of platelet recep-
tors for soluble agonists; (3) abnormalities of platelet
granules; (4) abnormalities of signal transduction path-
ways; (5) abnormalities of procoagulant phospholipids;
(6) miscellaneous abnormalities of platelet function,
which include inherited disorders of platelet function
that are less well characterized.
The diagnostic workup of patients with suspected
platelet function disorders should be done in specialized
centers following a two-step strategy. In the first step,
tests that are sensitive to most inherited platelet function
defects and that are not very expensive should be used.
LTA is certainly the gold standard among these tests: its
usefulness for detecting patients with platelet function
disorders has recently been confirmed in a prospective
study.26 However, traditional LTA is relatively insensi-
tive to the most common, mild platelet function disor-
ders, which are characterized by defects in platelet
granules or in the biochemical pathways that lead to
the secretion of the platelet granules contents. For
this reason, the use of lumiaggregometry to screen
these patients is preferable, as it also can easily detect
abnormalities of platelet secretion. At our center, we
use lumiaggregometry, morphologic examination of
164 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 35, NUMBER 2
peripheral blood smear, and clot retraction as the first
screening tests of platelet function disorders. In the
second step, specific diagnostic tests should be used to
confirm the diagnostic hypothesis that has been raised
based on the results of the screening tests used in the first
step. For instance, measurement of the platelet content of
adenine nucleotides and serotonin is the specific test to be
used to diagnose d-storage pool deficiency, and measure-
ment of the degree of inhibition of platelet adenylyl
cyclase activity by ADP is the test of choice to diagnose
defects of the platelet receptor for ADP, P2Y12.1
Abnormalities of the Platelet Receptors
for Adhesive Proteins
BERNARD-SOULIER SYNDROME
BSS is characterized by an autosomal recessive inher-
itance (one case only was characterized by autosomal
dominant inheritance), prolonged bleeding time, throm-
bocytopenia, giant platelets and decreased platelet sur-
vival, and quantitative or qualitative defects of the
platelet glycoprotein complex GPIb/IX/V.1 Typically,
BSS platelets do not agglutinate to ristocetin, and this
defect is not corrected by the addition of normal plasma.
The platelet aggregation responses to physiologic ago-
nists (ADP, collagen) are normal, except with low
concentrations of thrombin, because GPIba (one of
the two components of GPIb) plays a critical role in
the platelet aggregatory, secretory, and procoagulant
responses to thrombin.1
PLATELET-TYPE, OR PSEUDO, VON WILLEBRAND DISEASE
VWD is a disorder of primary hemostasis that is due to
the complete or partial defects of von Willebrand factor
(VWF), an adhesive protein that plays an essential role
in platelet adhesion and aggregation under high shear
forces. Platelet-type (or pseudo) VWD is not due to
defects of VWF, but to phenotypic gain of function of
the platelet GPIba, which has an increased avidity for
VWF, leading to the binding of the largest VWF
multimers to resting platelets and their clearance from
the circulation. The increased avidity of GPIba for
VWF accounts for the hyperresponse of platelet-type
VWD platelets to ristocetin, which can cause aggluti-
nation at very low concentration (0.5 mg/mL). Similar
results are obtained with PRP from patients with type
2B VWF, whose VWF has heightened affinity for
GPIba. The two disorders (i.e., type 2B VWD and
platelet-type VWD) can be differentiated using platelet
mixing studies.27
ABNORMALITIES OF GP IIB/IIIA
Glanzmann thrombasthenia (GT) is an autosomal
recessive disease that is caused by lack of expression
or qualitative defects of one of the two glycoproteins
2009
(GP IIB/IIIA) forming the integrin aIIb/b3, which in
activated platelets binds the adhesive glycoproteins that
bridge adjacent platelets, securing platelet aggrega-
tion.1 The diagnostic hallmark of the disease is the
lack or severe impairment of platelet aggregation in-
duced by any agonist; agonist-induced shape change of
GT platelets is normal. Ristocetin-induced agglutina-
tion of GT platelets is normal, although the maximal
increase in light transmission reached is reduced due
to defective, secondary, platelet-to-platelet contact-
induced aggregation.
ABNORMALITIES OF THE COLLAGEN RECEPTORS GP IA/IIA
(a2 b1) OR GP VI
Defects in both of these receptors are characterized by
selective impairment of platelet aggregation induced by
collagen. Typically, GP VI–deficient platelets are also
unresponsive to convulxin.1
Abnormalities of the Platelet Receptors
for Soluble Agonists
ABNORMALITIES OF THE THROMBOXANE A2 RECEPTORS
Platelets with defects of the TxA2 receptors do not
respond to arachidonic acid or to the thromboxane
receptor analogue U46619. The response to other ago-
nists may also be impaired, due to the lack of the
amplification of platelet aggregation and secretion that
is dependent on endogenous TxA2.1
ABNORMALITIES OF THE PLATELET a2-ADRENERGIC
RECEPTORS
Platelets lacking the a2-adrenergic receptors do not
undergo platelet aggregation upon stimulation with
epinephrine.1
ABNORMALITIES OF THE PLATELET P2Y12 RECEPTORS
Patients with P2Y12 deficiency have abnormalities of
platelet aggregation and secretion that are similar to
those observed in patients with defects of platelet
d-granules or other secretion defects (reversible aggre-
gation and no secretion in response to weak agonists
and impaired aggregation and secretion in response
other agonists, including collagen or thrombin). How-
ever, P2Y12 deficiency results in a more severe impair-
ment of the aggregation response to ADP: even at very
high concentrations (>10 mM), ADP elicits only a
slight and rapidly reversible platelet aggregation.1 In
contrast, platelets that undergo reversible aggregation
upon stimulation with low concentrations of ADP (2 to
4 mM), due to the presence of d-storage pool deficiency
or abnormalities of the biochemical pathways leading to
secretion of the granules contents, undergo full aggre-
gation upon stimulation with high concentrations of
ADP (Fig. 3).
 LIGHT TRANSMISSION AGGREGOMETRY/CATTANEO 165

Figure 3 Platelet aggregation in citrated PRP from a normal healthy volunteer, a patient with d-storage pool deficiency
(d-SPD), a patient with severe P2Y12 deficiency, and a patient with a primary secretion defect (PSD) that was induced by ADP at
the concentrations (mmol/L) indicated beside the aggregation curves (see text for further details). It must be noted that some
patients with d-SPD and PSD may display normal platelet aggregation also when stimulated by low to intermediate
concentrations of ADP (2 to 4 mmol/L).

Abnormalities of the Platelet Granules
ABNORMALITIES OF THE d-GRANULES
The term d-storage pool deficiency (d-SPD) defines an
inherited abnormality of platelets characterized by defi-
ciency of dense granules (or d-granules) in megakaryo-
cytes and platelets. The disorder can also be acquired.
Platelets from patients with d-SPD display abnormal
platelet aggregation induced by weak agonists: reversible
and monophasic waves of aggregation induced by ADP
(2 to 4 mM) or epinephrine and lack of aggregation
induced by low to moderate concentrations of collagen.1
However, this pattern of platelet aggregation is not the
pathologic hallmark of the disorder, as it is common to
other disorders, and, most importantly, it may not be
observed in many affected patients. Indeed, there is a
large variability in platelet aggregation in patients with
d-SPD, which has been well documented in a large study
of 106 patients, which showed that 25% of them had
normal aggregation responses, whereas only 33% had
aggregation tracings typical for d-SPD.28 Lumiaggreg-
ometry, which measures platelet aggregation and secre-
tion simultaneously, is therefore a more sensitive
technique than LTA for detecting d-SPD and, more
generally, platelet secretion defects.
GRAY PLATELET SYNDROME
Gray platelet syndrome (GPS) owes its name to the gray
appearance of the patient platelets in peripheral blood
smears as a consequence of the rarity of platelet granules.
Although there is no pathognomonic pattern of abnor-
malities of platelet aggregation, these patients tend to
have abnormal platelet secretion induced by ADP and/
or collagen and impaired thrombin-induced platelet
responses.1
QUEBEC PLATELET DISORDER
The Quebec platelet disorder is an autosomal dominant
qualitative platelet abnormality characterized by severe
posttraumatic bleeding complications that are unrespon-
sive to platelet transfusion, increased expression and
storage of urokinase plasminogen activator in platelets,
abnormal proteolysis of a-granule proteins, and reduced
to normal platelet counts. In some patients, platelet
aggregation induced by epinephrine is absent.29 Some
patients also have reduced aggregation with ADP and
collagen.29
Abnormalities of the Signal-Transduction
Pathways
Inherited abnormalities of the arachidonate/thrombox-
ane A2 pathway, which may be caused by defects of
phospholipase A2 (which liberates arachidonic acid from
membrane phospholipids), cyclooxygenase, or throm-
boxane synthetase, are associated with abnormalities of
the TxA2-dependent platelet aggregation and secretion;
the responses to stimulation by thromboxane analogues
are normal.
Other defects of signal transduction pathways
display miscellaneous abnormalities of platelet aggrega-
tion and secretion.1
166 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 35, NUMBER 2
Miscellaneous Abnormalities of Platelet
Function
PRIMARY SECRETION DEFECTS
These patients display abnormalities of platelet aggre-
gation and secretion that are similar to those observed in
patients with d-SPD or defects of the arachidonate
pathway. However, these patients have normal platelet
granule contents and normal TxA2 generation.1
CONCLUSION
Although it was first introduced almost 50 years ago,
LTA is still the most commonly used method to study
platelet function. LTA is a time-consuming and tech-
nically challenging technique that is affected by many
preanalytical and analytical variables, and these must be
carefully controlled for by expert personnel. For this
reason, LTA should be performed only in highly speci-
alized laboratories. LTA is useful in the initial screening
of patients with defects of platelet function. However, its
sensitivity to the most common inherited platelet func-
tion disorders, which are associated with abnormalities
of platelet secretion, is suboptimal. Lumiaggregometry,
which measures platelet aggregation and secretion
simultaneously, might be preferable to LTA for the
diagnostic workup of these patients.
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