Newer Platelet indices

Abstract
Thrombocytopenia is associated with bleeding risk. However, in thrombocytopenic patients, platelet count does
not correlate with bleeding risk and other factors are thus likely to contribute to this risk. This review presents
currently available platelet-related markers available on automated haematology analysers and commonly used
methods for testing platelet function. The test principles, advantages and disadvantages of each test are
described. We also evaluate the current literature regarding the clinical utility of the test for prediction of
bleeding in thrombocytopenia in haematological and oncological diseases. We find that several platelet-related
markers are available, but information about the clinical utility in thrombocytopenia is limited. Studies support
that mean platelet volume (MPV) can aid diagnosing the cause of thrombocytopenia and low MPV may be
associated with bleeding in thrombocytopenia. Flow cytometry, platelet aggregometry and platelet secretion
tests are used to diagnose specific platelet function defects. The flow cytometric activation marker P-selectin
and surface coverage by the Cone-and-Plate[let] analyser predict bleeding in selected thrombocytopenic
populations. To fully uncover the clinical utility of platelet-related tests, information about the prevalence of
platelet function defects in thrombocytopenic conditions is required. Finally, knowledge of the performance in
thrombocytopenic samples from patients is essential.
Platelets are essential in primary haemostasis, and it is evident that a low platelet count is a significant risk
factor for bleeding [1]. Thus, bleeding is a frequently occurring complication and may be the cause of death in
thrombocytopenic patients [2, 3], but not all patients with thrombocytopenia experience bleeding. In a study of
almost 30 000 platelet counts obtained from haematological and oncological patients, clinically significant
bleeding (WHO grade 2) was experienced on only 25% of days where platelet count was 5 109/L [3]. When
platelet count was between 6 and 80 109/L, the risk of bleeding was 17% and did not seem to correlate with
platelet count [3]. Likewise, there was no clear association between platelet count and bleeding found in other
studies among haematological or oncological patients [4, 5]. In other thrombocytopenic conditions such as liver
cirrhosis or sepsis, the relation between platelet count and bleeding has only been sparsely investigated [6, 7].
Thus, other factors obviously contribute to the risk of bleeding in thrombocytopenia. In this regard, the
haemostatic capacity of platelets depends on both number and function.
The aim of this review was therefore to describe the evidence for using platelet-related markers and methods
for testing platelet function in assessment of spontaneous bleeding risk in thrombocytopenic patients.

Automated haematology analysers

Principle
Platelet count obtained by automated analysers deviates due to differences in the applied methodology and
detection algorithms [8-10]. The commonly used impedance method detects cells by increase in electrical
impedance when a cell passes through an aperture in the flow cytometer. Platelets are hereby detected by cell

size because the increase in impedance is proportional to cell volume [11]. For optical light scatter method, cells
are also analysed based on volume when cells pass through a laser beam in the flow cytometer. If a two-angled
method is used, light is two-dimensional permitting additional analysis of cell granularity [11].
Hence, the commonly used impedance, but also the single-angle optical light scatter method, determines
platelet number by counting cells within a specific size range. To differentiate platelets from non-platelet
fragments and red blood cells, the analyser generates a (log-normal) distribution curve from the initial platelet
histogram (Table 1) [11-13]. Inaccuracies in the platelet count can occur if non-platelet particles with the same
size as platelets, for example microcytes, interfere with platelet counting obtained by methods relying on cell
size. This is critical in thrombocytopenic samples as the relative effect on the platelet count of misclassified cells
increases as the platelet count decreases [13]. Furthermore, large platelets or platelet clumps may not be
adequately identified due to the lack of distinction between red or white blood cells and platelets. This might
result in an underestimation of the platelet count [10, 13]. The advantages of these methods are that they
rapidly and at low cost measure platelet count. More accurate methods have been developed but are only
available on a few analysers. It includes an immunological method with application of a platelet-specific
antibody or fluorescent labelling of platelets prior to counting in the flow cytometer [11]. Previously, also manual
counting by phase contrast microscopy was often used, but this method is time-consuming and imprecise [9].
Name of test

Principle

Advantages

Disadvantages

Clinical utility

1. CV, coefficient of variation; ITP, immune thrombocytopenia.

Platelet indices
Low accuracy and high CV
% at low platelet count

Platelet count

Automated flow cytometric

Simple, rapid

analysis, most often impedance

Low volume

Dependent on
methodology, risk of
interference or lack of
detection of large platelets

Diagnostic for thrombocyt

No correlation with bleedin

in thrombocytopenic patien
general [3-5]

or platelet clumps

High predictive value for b

Mean platelet

Derived from the platelet

volume, MPV

distribution curve

Simple, rapid
Low volume
Widely available

marrow failure as a cause o

Dependent on the platelet

thrombocytopenia [15, 25,

distribution curve

Low MPV may be associa

bleeding when platelet cou

<20 109/L [24]

Platelet
distribution
width, PDW

Derived from the platelet

Simple, rapid

Dependent on the platelet

No information in relation

distribution curve

Low volume

distribution curve

bleeding risk in thrombocy

Name of test

Principle

Advantages

Disadvantages

Clinical utility

1. CV, coefficient of variation; ITP, immune thrombocytopenia.

Fraction of large

Derived from the platelet

Simple, rapid

Dependent on the platelet

No information in relation

cells

distribution curve

Low volume

distribution curve

bleeding risk in thrombocy

Simple, rapid,
Immature platelet

Staining of RNA in platelets (flow

low volume

fraction, IPF

cytometry)

No interference

A measure of thrombopoie
Limited availability

bleeding risk in thrombocy

from other cells

Mean platelet

Refractive index obtained by optical

Simple, rapid

component, MPC

light scatter (flow cytometry)

Low volume

No information in relation

Limited experience

No information in relation

bleeding risk in thrombocy

Methods for platelet function testing

Time-consuming
Light
transmission
aggregometry

Require experienced

Diagnostic for platelet surf

Change in turbidity due to platelet

Gold standard

operator, sample

glycoprotein defects

aggregation in response to agonists

Widely used

preparation and large blood

No information on bleedin

volume

thrombocytopenia

High CV%

Whole blood
aggregometry

Changes in impedance due to

platelet aggregation to electrodes in
response to agonists

Whole blood
Available as a
point-of-care

Diagnostic for platelet surf

High CV%

test

glycoprotein defects

No information on bleedin
thrombocytopenia

(i) Lumiaggregometry:
Light transmission aggregometry or
whole blood aggregometry
Platelet secretion
assays

combined with bioluminescence

High sensitivity

For solitary assays, sample

assay for detection of dense granule

for platelet

preparation is required and

release

secretion defects

no common standard exists

Fluorescently labelled antibodies,

Whole blood,

Expensive

Diagnostic for platelet surf

cell size and granularity by light

small volume

Require specialised

glycoprotein defects or pla

scatter

Wide variety of

operator

secretion defects

Diagnostic for platelet secr

defects

(ii) Solitary assays: Detection of

released substances or granule
content
Flow cytometry

tests

Low platelet activation (lo

Name of test

Principle

Advantages

Disadvantages

Clinical utility

1. CV, coefficient of variation; ITP, immune thrombocytopenia.

Not dependent

selectin) predicts bleeding

on platelet count

myeloid leukaemia [62]

Low CV%
Whole blood

Invasive

Not recommended for scre

Bleeding time ad

Cessation of blood flow in vivo after

In vivo test

High CV%

for platelet function defect

modum Ivy

a standardised cut in skin

Point-of-care

Low sensitivity for platelet

No information on bleedin

test

secretion defects

thrombocytopenia

Platelet function
analyser-100,
PFA-100

Whole blood,
High-shear platelet adhesion and

rapid, simple

Low sensitivity for platelet

plug formation

Point-of-care

secretion defects

test

Cone-andHigh-shear platelet adhesion and

rapid, simple

analyser (Impact-

aggregation onto surface

Point-of-care

R)

No information on bleedin
thrombocytopenia

Not for diagnostic use

Whole blood,

Plate[let]

Screening test

Low response is associated

Little experience

bleeding [63, 74] and corre

with severity of bleeding

test

symptoms in ITP [63]

Whole blood,

Platelet function is not

Viscoelastic

Dynamic change in viscoelastic

rapid, simple

directly tested in the

method

properties due to clotting

Point-of-care

standard assay but depends

test

on thrombin generation

Not for diagnostic use

Low response associated w

bleeding [79]

Table 1. Characteristics and clinical utility of platelet markers

Overall, automated haematology analysers overestimate platelet count in thrombocytopenic samples compared
with the immunological reference method [8, 9]. The coefficient of variation (CV) increases as platelet count
decreases [8, 9]. In a recent survey based on United Kingdom National External Quality Assessment Scheme,
CV% reached 1543% when platelet count was below 10 109/L [9].
Other platelet indices can be derived from the platelet distribution curve obtained from impedance or optical
methods. It includes mean platelet volume (MPV), platelet distribution width (PDW) and the fraction of large
platelets. The MPV describes the average platelet size reported in femtolitre (fL) and is available on most
haematology analysers [11, 12, 14]. The PDW is a measure of the heterogeneity in platelet size either defined
as the distribution width at 20% frequency level or calculated as the standard deviation of platelet volume
divided by MPV 100 [12]. Derived platelet indices are, however, highly specific to the individual technologies,
with different analysers having different reference ranges [12]. As a separate feature, the fraction of large
platelets can be addressed specifically: On Sysmex analysers (Sysmex, Kobe, Japan), the parameter is

called platelet large cell ratio (P-LCR) and is the percentage of platelets larger than 12 fL [11, 12]. On the
ADVIA analyser (Siemens Healthcare, Erlangen, Germany), the ratio of platelets higher than 20 fL is
reported [11].
Factors that affect platelet counting (interference from cells or cell fragments, inadequate detection of large
platelets or platelet clumps) also influence platelet indices that are calculated from the platelet distribution curve.
If red blood cells are misclassified as platelets, it causes an overestimation of MPV, a higher PDW and an
increase in fraction of large cells. Moreover, in severe thrombocytopenia, it may not be possible to obtain a
sufficient platelet distribution curve to calculate other platelet indices than the platelet count [12].
There have been special concerns about the recommended anticoagulant for platelet counting, K2 or
K3 ethylenediaminetetraacetic acid (EDTA), because it affects MPV. EDTA causes an increase in MPV from
7.9% within 30 min to 13.4% over 24 h when measured by impedance [15] and decreases by 10% when
determined by an optical method [12, 14]. Time delay probably also affects other variables, for example PDW
and the fraction of large cells [14]. Therefore, it is recommended to process the sample within 120 min when
platelet indices are determined [16]. EDTA may also cause agglutination of platelets, resulting in a falsely low
platelet count (pseudo-thrombocytopenia). Each laboratory should, however, have strategies to detect spurious
low platelet counts [13].
Reticulated platelets can be determined by flow cytometry with a fluorescent dye that selectively binds RNA.
Assessment of reticulated platelets is available only for Sysmex haematology analysers and is called
immature platelet fraction (IPF). The IPF is measured in a dedicated platelet chamber and is given as a
percentage of the total platelet count or as an absolute number [12]. Immature platelet fraction is not affected by
the above-mentioned methodological issues and is stable for 48 h [17]. Finally, the ADVIA analyser reports a
mean platelet component (MPC) based on the refractive index using an optical method [11]. It corresponds to
platelet density, and a decrease in density suggests platelet activation [18].

Clinical utility of parameters related to platelet size

Large platelets have higher platelet granule content and greater extent of secretion, membrane protein
activation and platelet aggregationin vitro [19, 20]. Several clinical studies have documented an association
between haemostasis and platelet size as they found MPV to be an independent risk factor for thrombosis [21],
and large platelets are selectively consumed during massive bleeding [22, 23]. However, only few studies have
investigated the relationship between MPV and spontaneous bleeding in thrombocytopenia, and no data on
MPC, PDW or the fraction of large platelets have been published.
Eldor et al. [24] investigated 175 haematological patients with platelet counts below 20 109/L. They found that
MPV was better than the platelet count for identifying bleeding phenotype. Unfortunately, it was not stated
whether MPV was evaluated during active bleeding. If so, bleeding could possibly have affected the MPV and
thus the result of the study. The study indicated that platelet mass (platelet count MPV) might be associated
with platelet haemostatic capacity. The importance of platelet mass is supported by the finding that platelet
count and MPV are heritable and regulated traits and the platelet mass is relatively constant in healthy
populations [20, 25]. Van der Lelie et al. [26] did, however, not find an association between MPV and bleeding in

patients with platelet count below 50 109/L in a relatively small study (n = 44) that included thrombocytopenia
of various causes.
Several clinical studies have shown that MPV and PDW are lower when thrombocytopenia is caused by
reduced production (e.g. leukaemia or chemotherapy-induced) compared with peripheral destruction as seen in
patients with immune thrombocytopenia (ITP). The predictive value of MPV for bone marrow failure was 73% to
100% depending on the cut-off point used [15, 25, 27-31]. It has been postulated that the bleeding risk is higher
in acute leukaemia than in ITP [4, 32], and one may speculate whether it is partly caused by the documented
differences in platelet size. For P-LCR, the diagnostic value for bone marrow failure is unclear [27, 29, 33].

Clinical utility of immature platelet fraction

Newly released platelets contain RNA and progressively degranulate over the first 2436 h [34]. Thus, active
thrombopoiesis generates reticulated platelets, which increases IPF% [17]. The absolute immature platelet
count may, however, still be within reference range [17]. Hence, in patients with bone marrow suppression, an
increase in IPF% reflects marrow restitution [35]. The association between IPF% and haemorrhagic diathesis in
thrombocytopenia is unexamined, but in vitro newly released platelets seem more haemostatically active than
older platelets [36-40]. A high IPF indicates bone marrow compensation and possibly a low risk of bleeding.
Accordingly, IPF% is high when thrombocytopenia is due to peripheral destruction of platelets as in ITP or
thrombotic thrombocytopenic purpura [17, 41], which are conditions associated with relatively low risk of
bleeding [32].

Summary on platelet markers on automated haematology analysers

Platelet indices are available on automated haematology analysers and are easily addressed along with the
platelet count using standard EDTA samples. Only it is advisable to perform the analysis within 120 min from
withdrawal. The total platelet mass may superiorly indicate haemostatic capability compared with measurement
of platelet count alone. Published evidence supports the aid from MPV when diagnosing the cause of
thrombocytopenia (reduced production vs. increased destruction of platelets). The reference range of MPV is
specific to the individual technologies. Thus, a clear cut-off cannot yet be provided.

Methods for measuring platelet function

In the following section, we address platelet function tests with focus on methods that are already widely used in
patients with bleeding tendency. We excluded methods primarily used for monitoring the efficacy of platelet
inhibitors. For that reason, PlateletWorks and VerifyNow are not mentioned. The platelet function tests are
summarised in Table 1.

General aspects
Platelets are fragile and are easily activated. The blood sample shall therefore be obtained by an experienced
phlebotomist using minimal tourniquet pressure [42]. The sample should be mixed gently and be kept at room
temperature before processing. Analysis is recommended to be carried out within 120 min [42]. Tests address

different aspects of platelet function (adhesion, activation and aggregation), and all tests are unfortunately yet
poorly standardised [43]. They are performed on either platelet-rich plasma or whole blood, but whole-bloodbased analyses may be advantageous as they comprise a more physiological test matrix.

Specific tests
Platelet aggregometry.
Principle: Aggregometry determines platelet aggregation after addition of agonists for specific platelet
membrane receptors. In light transmission aggregometry, platelet aggregation is detected as an increase in
turbidity due to platelet clumping in platelet-rich plasma with reference to platelet-poor plasma [44]. In whole
blood aggregometry, platelet aggregation is measured as an increase in impedance in whole blood when
platelets aggregate to two electrodes in the solution [45].
The execution of light transmission aggregometry requires trained personnel and is laborious as well as costly.
It also requires a relatively large blood volume [44-46]. A potential clinically applicable point-of-care device for
measurement of whole blood aggregation, the Multiplate Analyser, has been introduced. The system is simple
to operate and produces results quickly [47]. Platelet count is a major determinant for aggregometry results
even at platelet counts within reference range [46, 48].
Clinical utility: Light transmission aggregometry is the gold standard for diagnosing platelet membrane
glycoprotein defects such as BernardSoulier syndrome and Glanzmann's thrombasthenia, both known to be
associated with severe bleeding [42, 49]. As described in the next section, aggregometry also detects platelet
secretion defects, defined as diminished granule content or affected granule secretion. Data on the diagnostic
value of Multiplate are limited, but it is sensitive to Glanzmann's thrombasthenia [50, 51]. Information is lacking
about the relationship between aggregometry and acquired thrombocytopenic conditions or spontaneous
bleeding. Impaired aggregometry results are, however, common in non-thrombocytopenic patients with a
positive bleeding history with few false positives in persons without bleeding problems [52].
Platelet secretion assays.
Principle: By light transmission or whole blood aggregometry, the platelet secretion from dense granules is
evaluated indirectly. The applied agonist causes release of platelet granules, the content of which activates
other platelets in suspension and amplifies aggregation response. The sensitivity to platelet secretion defects is
lower compared with direct assessment of granule release using a lumiaggregometer or solitary assays [49]. By
lumiaggregometry, a bioluminescent assay is applied for direct measurement of platelet secretion from dense
granules in parallel with aggregation response. Briefly, luciferin reacts with ATP released from platelet granules.
The reaction is catalysed by luciferase and results in generation of light that is measured [44, 53]. Solitary
assessment of dense granule release is performed by, for example, high-pressure liquid chromatography,
enzyme-linked immunosorbent assay (ELISA) or flow cytometry. The principle is often bioluminescence or
based on detection of serotonin release from dense granules [42, 53]. Platelet alpha granule proteins, for
example platelet factor 4 (PF4) and beta-thromboglobulin (bTG), can be measured by ELISA,
radioimmunoassay or Western blotting[42].
An abnormal result when evaluating granule release may be caused by a low granule number or aberration in
the mechanisms governing granule secretion. The granule number can be investigated directly either by

electron microscopy or by measuring the total granule content in lysed platelet preparations [42]. For some
methods, results are reported adjusted for platelet count [42]. An obvious challenge is the availability of a wide
variety of assays with different test principles.
Clinical utility: There is a lack of consensus regarding the diagnosis of platelet secretion defects, and no
systematic evaluation of the prevalence in thrombocytopenic conditions has been performed. Platelet secretion
defects can occur as acquired conditions and may therefore develop as part of the pathophysiological process
in thrombocytopenia [49, 54, 55]. This is especially likely in conditions affecting the bone marrow directly such
as leukaemia where platelet secretion defects in fact have been reported [56, 57]. Platelet granule defects
actually seem to be relatively common in pathological states, for example myelodysplastic syndrome [55].
Platelet secretion defects may therefore potentially contribute to bleeding risk in thrombocytopenia [58].
Flow cytometry.
Principle: Flow cytometry applies fluorescently conjugated antibodies to detect surface antigens on cells in
suspension. Thus, the number of surface epitopes for the antibody can be quantified. In addition, the cells are
categorised according to their light scatter properties generally reflecting the size and granularity of the cell [47,
59]. Platelet surface antigens can be investigated with platelets in resting state and after application of an
agonist that activates the platelets. Hence, the platelet function is addressed by evaluation of a spectrum of
activation-dependent changes. A frequently used marker is P-selectin. P-selectin is translocated from
intracellular alpha granule membranes to the surface membrane upon platelet degranulation and therefore
indicates the presence of activated platelets [59, 60]. Dense granules can be evaluated by determining uptake
of the fluorescent dye mepacrine [60]. In addition, platelet interaction with other blood cells and plasma
components can be determined [47, 59]. It is also possible to measure platelet procoagulant activity and
platelet-derived microparticles [42].
An advantage of flow cytometry is that it rapidly measures specific characteristics of a large number of
individual cells in suspension and is independent of the platelet count [59]. The analysis requires only a small
volume of whole blood. However, the equipment is expensive, and the need for relatively complicated sample
preparation is a limitation to its use in a daily clinical practice [59].
Clinical utility: Flow cytometry detects platelet membrane glycoprotein defects and dense granule
deficiency [42, 60] and may assist in diagnosing ITP [59]: recently, it was found that ITP patients with bleeding
had lower content of platelet surface glycoprotein and less response after agonist application compared with
ITP patients who did not experience bleeding. The opposite was found in patients with acute myeloid leukaemia
or myelodysplastic syndrome [61]. The explanation is not clear, but ITP patients with bleeding may already have
experienced in vivo activation due to antiplatelet antibodies and therefore exhibited lower responsiveness when
tested. Alternatively, the primary haemostasis in acute myeloid leukaemia/myelodysplastic syndrome may be
dysregulated, causing platelet function defects [61].
Leinoe et al. [62] investigated predictors of bleeding within 28 d from the time of diagnosis or first relapse in a
cohort study of 50 patients with acute myeloid leukaemia. Patients with bleeding had lower platelet count and
lower P-selectin expression after in vitro platelet activation. Low P-selectin surface membrane expression
predicted risk of bleeding within 28 d in univariate analysis [62]. P-selectin is only present on the surface for a

short period after activation [60]. Therefore, blood sampling at the time of bleeding causes low measures of Pselectin if in vivo platelet activation has already occurred. Alternatively, the low P-selectin expression may be
caused by an acquired platelet secretion defect. In the above-mentioned study by Panzer et al. [63], the Pselectin surface expression did not correlate with the bleeding score in ITP patients.
Bleeding time and Platelet function analyzer-100.
Principle: Template bleeding time is the time taken for bleeding to stop after a standardised cut into the
skin [64]. The template bleeding time is invasive and time-consuming to perform and is dependent on variables
not related to haemostasis such as skin thickness and temperature [42].
The Platelet function analyzer-100 (PFA-100; Siemens Healthcare Diagnostics GmBH, Marburg, Germany) is a
device where whole blood is aspirated at high shear rate through an aperture coated with prespecified
agonists [65, 66]. The agonists cause platelet adhesion, activation and aggregation. The result is given as time
to occlusion of the aperture due to platelet plug formation [65]. The advantage of this method is that it uses
whole blood and is rapid to perform with the use of disposable test cartridges [66]. Closure time increases when
platelet count decreases. If the aperture is not closed after 300 s, the apparatus reports non-closure. Nonclosure is increasingly reported at platelet counts below 30 109/L, and virtually, all tests result in non-closure at
platelet count below 10 109/L [65, 67].
Clinical utility: Template bleeding time and PFA-100 cannot be used for the diagnosis of specific platelet
function defects [42], but are sensitive to severe platelet membrane receptor dysfunctions [53, 66]. Template
bleeding time is invasive and has low sensitivity and specificity for platelet secretion defects and poor
reproducibility [42]. It is therefore no longer recommended for the evaluation of platelet function defects [42, 49,
66].
It has not been specifically addressed whether template bleeding time or PFA-100 closure time correlates with
bleeding risk in thrombocytopenia. PFA-100 is unlikely to contribute with additional information because it has
no discriminative value at low platelet counts (reporting non-closure). Furthermore, PFA-100 closure time does
not correlate with the presence or severity of bleeding symptoms in platelet secretion defects [66, 68].
Cone-and-Plate[let] analyser (Impact-R).
Principle: In the Cone-and-Plate[let] analyser, whole blood is placed on a well-defined surface and a shear rate
is applied. In the commercialised version (Impact-R; Matis Medicals Inc, Beersel, Belgium), a polystyrene
surface is used. The surface induces platelet adhesion and subsequent secretion and aggregation. Afterwards,
bound platelets are stained and measured using an image analyser. The result is given as the percentage of
surface covered with stained platelets and the average size of the platelet aggregates retained [69]. A positive
correlation exists between platelet count and surface coverage or average size of platelet aggregate [63, 69].
An exception is that average size of platelet aggregate was found not to correlate with platelet count in patients
with ITP [63]. The process is automated and provides a result within 6 min. It is easy to perform and requires
only a small quantity of blood [47].
Clinical utility: The Cone-and-Plate[let] analyser performs an overall evaluation of the platelet adhesion and
subsequent secretion and aggregation process. The test result is unspecific and cannot be used for direct
diagnostic purposes. Overall, the clinical applicability of the method has only been sparsely investigated. The

surface coverage is found to be decreased in patients with known BernardSoulier syndrome [70], Glanzmann's
thrombasthenia [69] or platelet secretion defects [71]. Increased surface coverage is seen in patients with
thrombotic thrombocytopenic purpura [72]. Without regarding the platelet count, the analyser has shown high
predictive value for excluding a bleeding disorder in one study [73]. Moreover, Kenet et al. [74] studied 42
thrombocytopenic patients from a haematological department. They found that patients with bleeding symptoms
had lower percentage of surface coverage and lower average size of aggregates compared with patients
without bleeding symptoms. Bleeders also had lower platelet count and MPV. When taken together, 95%
(21/22) of patients with low surface coverage (<5%) and low MPV had bleeding symptoms [74].
Panzer et al. [63] investigated bleeding symptoms based on clinical investigation and bleeding history in 41 ITP
patients. Platelet count and surface coverage, but not the average coverage, were associated with bleeding
symptoms. Furthermore, they found that P-selectin expression did not correlate with bleeding symptoms[63].
Viscoelastic methods.
Principle: The tests thromboelastography (TEG; Haemoscope Corporation, Niles, IL, USA) and
thromboelastometry (ROTEM, Pentapharm, Munich, Germany) are used to monitor the coagulation process in
whole blood [75]. The result is a curve describing the dynamic changes in viscoelastic properties due to clotting
of the sample. The analysis provides information on clot initiation, clot formation and clot strength [75]. Also, an
assay for specifically testing platelet function (PlateletMapping, Haemoscope Corporation, Niles, IL, USA) is
available when using the TEG [76]. The clot strength is highly dependent on platelet count, but the detection
threshold is unknown [75]. The test provides rapid results and is easy to perform.
Clinical utility: Viscoelastic tests evaluate global haemostasis besides the interaction with the vessel wall.
Hence, they address platelet activation and aggregation, but not adherence. Distinct platelet function defects
cannot be diagnosed using these tests, but clot strength is decreased in patients with Glanzmann's
thrombasthenia [77, 78]. When using specific platelet agonists (Platelet Mapping), the clot strength is also
diminished in BernardSoulier syndrome [78].
Curves from viscoelastic methods are currently used during peri- or post-operative bleeding to promptly
diagnose the coagulopathy and to provide targeted treatment [75]. Gunduz et al. [79] found a diminished clot
strength in 39 patients with ITP while experiencing mild bleeding symptoms. The ITP patients with bleeding also
had lower platelet count, which may in itself explain the lower clot strength [79].

Summary on methods for measuring platelet function

An increased knowledge on the prevalence of platelet function defects is the basis for determining the value
and extent of platelet function testing in thrombocytopenia. In this regard, flow cytometry is particularly
advantageous because it provides information on platelet pathophysiology even in severely thrombocytopenic
samples. It is therefore suitable for descriptive studies in thrombocytopenia with pursue of better diagnostics
and distinction between different phenotypes. Flow cytometry is already increasingly being used for diagnosing
known platelet defects.
The template bleeding time and PFA-100 have limitations, including lack of sensitivity for presently known
platelet secretion defects. The remaining methods detect functional platelet defects although the evidence is

sparse for some tests. Current evidence suggests that the flow cytometric marker of activation P-selectin and
surface coverage by the Cone-and-Plate[let] analyser predict bleeding in selected thrombocytopenic
populations (have high sensitivity). A test that adequately rules out bleeding (high specificity) would also be of
clinical relevance.
Besides flow cytometry and some secretion assays, low platelet count independently reduces the response and
thus the ability to diagnose distinct platelet function defects. The total haemostatic capacity of platelets must
depend on both platelet number and function. Therefore, results may still potentially predict clinical outcome. In
this regard, the knowledge of the detection threshold and CV when testing samples with low platelet count is
essential but is for most methods unknown. It is an advantage that methods are rapid, accurate and easy to
perform, which holds true for Multiplate (Roche Diagnostics GmbH, Mannheim, Germany), Cone-and-Plate[let]
analyser viscoelastic methods and PFA-100.

Conclusion and perspectives

Platelet count can be used to identify a group with an a priori high risk of bleeding, but additional markers are
needed for further stratifying patients into those with low and high risk of bleeding.
Haemostasis depends on platelets but also other factors such as the integrity of the vessel wall, von Willebrand
factor for adhesion and secondary haemostasis for fibrin polymerisation and consolidation of the platelet plug.
Impairment of other parts of haemostasis has been described in thrombocytopenia. The present review has,
however, focused on platelet-related markers, and issues can be pointed out:

Clinical practice
1.

Several tests (platelet indices, Multiplate, Cone-and-Plate[let] analyser, viscoelastic methods and
PFA-100) are rapid and easy to perform, and without regarding the indication for the test, they are
therefore highly suitable for point-of-care testing.

2.

MPV can aid in diagnosing the cause of thrombocytopenia, but a clear cut-off cannot yet be provided.

3.

Some platelet function tests (flow cytometry, aggregometry and platelet secretion tests) are presently
used for diagnosing platelet function defects.

Research agenda
1.

Validation of methods that determine platelet function in samples with low platelet count, including
evaluation of detection threshold and CV%.

2.

Further standardisation of platelet function tests.

3.

Investigation of markers of clinical outcome including predictive value for bleeding.

4.

Descriptive studies of distinct platelet phenotypes in thrombocytopenic condition to clarify the

prevalence of platelet function defects.