Evaluation of a whole blood remote platelet function test for the diagnosis of mild bleeding disorders

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/jth.12555 This article is protected by copyright. All rights reserved.

Received Date : 08-Nov-2013

Accepted Date : 23-Feb-2014

Article type : Brief Report - Clinical Haemostasis and Thrombosis

Evaluation of a whole blood remote platelet function test for the diagnosis of mild bleeding

disorders

Short title: Remote platelet test for bleeding disorders

Natalia Dovlatova*,§, Marie Lordkipanidzé§, Gillian C. Lowe§, Ban Dawood§, Jane May*, Stan

Heptinstall*, Steve P. Watson§ and Susan C. Fox*, for the UK GAPP Study Group

*Cardiovascular Medicine, University of Nottingham, Queens Medical Centre, Nottingham, United

Kingdom

§Centre for Cardiovascular Sciences, Birmingham Platelet Group, University of Birmingham,

Birmingham, United Kingdom

Institution: The University of Nottingham and The University of Birmingham

Address for correspondence:

Natalia Dovlatova

Cardiovascular Medicine

D Floor, South Block

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This article is protected by copyright. All rights reserved.

Queen’s Medical Centre

Nottingham

NG7 2UH

United Kingdom

Tel +44 (0)115 8231019

Fax +44 (0)115 8231017

E-mail [email protected]

Abstract

Background: Mild platelet function disorders (PFDs) are complex and difficult to diagnose. The

current gold standard test, light transmission aggregometry (LTA), including lumi-aggregometry, is

time- and labour-intensive and blood samples must be processed within a limited time after

venepuncture. Furthermore, many subjects with suspected PFDs do not show a platelet

abnormality on LTA.

Objective: To assess the diagnostic potential of an easy-to-use remote platelet function test

(RPFT) as a diagnostic pre-test for suspected PFDs.

Methods: RPFT was compared to lumi-aggregometry in participants recruited to the Genotyping

and Phenotyping of Platelets study (GAPP, ISRCTN 77951167). For RPFT, whole blood was

stimulated with platelet agonists, stabilized with PAMFix and returned to the central laboratory

for analysis of P-selectin and CD63 by flow cytometry.

Results: In the 61 study participants (42 index cases and 19 relatives) there was a good agreement

between lumi-aggregometry and RPFT with diagnosis being concordant in 84% of cases

(kappa=0.668, p<0.0001). According to both tests, 29 participants were identified to have a

deficiency in platelet function and 22 participants appeared normal. There were 4 participants

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where lumi-aggregometry revealed a defect but RPFT did not, and 6 participants where RPFT

detected an abnormal platelet response that was not identified by lumi-aggregometry.

Conclusion: This study suggests that RPFT could be an easy-to-use pre-test to select, which

participants with bleeding disorders would benefit from extensive platelet phenotyping. Further

development and evaluation of the test are warranted in a wider population of patients with

excessive bleeding and could provide informative screening tests for PFDs.

Keywords: bleeding, blood platelet disorders, platelet function tests, flow cytometry, platelets

Introduction

Mild platelet function disorders (PFDs) are complex, poorly understood, difficult to diagnose and

require extensive laboratory testing limited to specialised laboratories [1,2]. Lumi-aggregometry,

where dense granule secretion is assessed in parallel with traditional LTA [3-5] is informative, but

is time- and labour-intensive. It is performed on platelet-rich plasma and requires a considerable

volume of a fresh blood sample. In our previous study we demonstrated using lumi-aggregometry

that approximately 60% of participants with excessive clinical bleeding and a suspected

underlying inherited PFD have a demonstrable abnormality in platelet responses [5]. This suggests

that 40% of patients may have a platelet abnormality that is not detectable by lumi-aggregometry

or may have a defect in other components of the haemostatic pathway or a combination of mild

defects, which leads to a bleeding phenotype. These findings are in agreement with an earlier

study by Quiroga et al where approximately 60% of a slightly different patient population with

mild bleeding had no identified defect when assessed by LTA and serotonin and ATP release

measurements [6]. Accordingly, a simple and easy-to-perform preliminary test might be useful to

select patients with excessive bleeding for whom extensive platelet phenotyping using traditional

methodologies may be required for further characterisation. At present, there is no reliable easy-

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to-use and sufficiently sensitive global platelet function test available for use as a screening tool in

subjects with suspected PFD [7].

We have developed a remote platelet function test (RPFT), in which a blood sample is

manipulated in a simple way that does not require specialised staff or equipment at the test site.

The sample is stabilised utilising a fixing solution, PAMFix, and sent to a flow cytometry fascility

for analysis [8], where the expression of the α-granule marker P-selectin and the dense granule

marker CD63 is measured. The samples treated with PAMFix are stable for up to 9 days at

ambient temperature. CD63 expression specifically evaluates dense granule secretion; analysis of

P-selectin expression is used as a marker of α-granule secretion and also as a general indicator of

platelet reactivity. Here we describe the evaluation of this new approach against traditional lumi-

aggregometry in patients with suspected inherited PFDs.

Methods

Study population

To assess the diagnostic potential of the RPFT in the diagnosis of PFDs, results were compared to

lumi-aggregometry in participants recruited to the Genotyping and Phenotyping of Platelets study

(GAPP, ISRCTN 77951167) from April 2012 to March 2013. We studied 61 participants (42 index

cases and 19 recruited relatives), 13 males (median age 13, range 5-45) and 48 females (median

age 39, range 2-77); all had a history of excessive bleeding with a suspected underlying inherited

PFD. These participants had normal coagulation parameters and a normal platelet count and were

referred from UK Comprehensive Care Haemophilia Centres. Patients with an existing diagnosis of

a known platelet defect were not included in this study. Bleeding symptoms in adult patients

(n=42) were assessed using the ISTH/SSC bleeding assessment tool (BAT) [9]. All the participants

presented with a significant bleeding history with a median bleeding score of 13 (interquartile

range: 8 – 16), whereas the 95th percentile within the healthy volunteer population was 4 [10].

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Platelet function was also assessed in 41 healthy volunteers recruited as controls, who had

no history of bleeding and had refrained from taking any medication known to affect platelet

function. All blood samples were either taken at or delivered by courier from several referring

centres (maximum distance 100 miles) to the central laboratory as previously described [5]. This

study was approved by the National Research Ethics Service Committee West Midlands –

Edgbaston (ref 06/MRE07/36). All participants provided written informed consent.

Assessment of platelet function

For the RPFT measurements, whole blood samples collected into sodium citrate were prewarmed

and stimulated at 37oC for 5 min without mixing. They were then fixed using PAMFix (Platelet

Solutions Ltd, Nottingham). Samples were then transported from the central laboratory to the

flow cytometry facility (60 miles) and routinely analysed within 3 days. For platelet stimulation

blood samples were treated with 1) saline alone to provide a baseline test; 2) adenosine

diphosphate (ADP) with U46619, a stable thromboxane A2 analogue (ADP/U4 10μM and 1μM

respectively); 3) thrombin receptor activating peptide (TRAP [SFLLRN] 20μM) and 4) arachidonic

acid with epinephrine (AA/EPI, 0.5mM and 100μM respectively). U46619 and epinephrine were

used as potentiating agents and were chosen based on previous studies where we investigated

optimal conditions for platelet activation in whole blood [11]. Intra- and inter-assay coefficients of

variation are 4.4% and 5.6% respectively.

Lumi-aggregometry was performed as previously described [5]. The results were considered

abnormal when they fell below the 5th percentile with reference to a bank of local healthy

volunteers (n=68).

Statistical analysis

Results of platelet function measurements are presented as individual data in median

fluorescence units (mf). To determine the ability of the RPFT to discriminate between subjects

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with and without abnormal platelet responses and to set optimal cut-off levels, receiver operator

characteristic (ROC) curve analysis was performed. For P-selectin measurements ROC curves were

generated using the subjects with and without any defect, as determined by lumi-aggregometry,

and for CD63 measurements, subjects with and without a secretion defect. Agreement between

the two tests was assessed using two-by-two analysis and Cohen’s kappa statistic. Analyses were

performed using the software packages SPSS 14.0 and GraphPad Prism 6.

Results

According to lumi-aggregometry, 33 of the 61 participants recruited to GAPP study were

considered to have an abnormality in platelet responses. The majority of these 33 participants

could be subdivided into three categories on the basis of their lumiaggregation profile [5] –

defects in dense granule secretion (n=9), Gi signalling (n=14) and the TxA2 pathway (n=3). The

remaining 7 participants had either a complex defect that was difficult to assign to any specific

group, presenting with reduced responses via several pathways (n=4), or a defect in the response

to collagen (n=1) or TRAP (n=2).

The area under the ROC curve for P-selectin (Figure 1a) and CD63 measurements (Figure 1b) with

different agonists varied between 0.72 and 0.99 (p<0.004). This confirmed that RPFT can

discriminate between individuals with excessive bleeding with and without abnormal platelet

responses as detected by lumi-aggregometry. Based on the ROC analysis, appropriate cut-off

levels for each parameter were chosen to provide the highest possible sensitivity of the RPFT to

minimise false negative results. Applying the chosen cut-offs in the group of healthy controls

indicated that 5 out of 41 participants presented with somewhat reduced platelet responses on

RPFT (Figure 2a). This small degree of overlap in platelet responsiveness between populations of

patients and healthy controls is also seen in other platelet function tests in addition to other tests

of haemostasis [6].

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In the patient group, the overall agreement between lumi-aggregometry and RPFT was

good, with the diagnosis being concordant in 84% of cases (kappa=0.668, p<0.0001) (Table). All

participants who presented with a dense granule secretion defect (Figure 2b) or a defect in the

TxA2 pathway (Figure 2c) on lumi-aggregometry were also classified as having a platelet

abnormality on the RPFT with at least one parameter falling below the normal range. Out of the

14 participants who presented with a defect in Gi signalling based on lumi-aggregometry (Figure

2d), 11 also showed abnormal platelet responses on RPFT, while 3 subjects were considered

normal. It should be noted that presentation of the Gi-type platelet defect on lumi-aggregometry

is variable, overlaps considerably with corresponding measurements in healthy volunteers (Figure

3) and is based on interpretation of kinetic information not available in endpoint assays. The

remaining case that was missed on RPFT presented with a reduced response to collagen on lumi-

aggregometry and was considered to have a potential defect in GPVI.

Six participants with normal responses on lumi-aggregometry presented with an abnormal

response on RPFT suggesting that this test may have an increased sensitivity. One of these

patients had normal aggregation and dense granule secretion on both tests, but markedly

reduced P-selectin expression in response to all agonists, indicative of impaired α-granule

secretion.

Discussion

Here we evaluated the performance of a novel remote platelet function test alongside lumi-

aggregometry in patients presenting with bleeding symptoms suggestive of an inherited PFD. The

new test directly assesses both α- and dense granule secretion in response to platelet stimulation

with several agonists in whole blood. The RPFT was able to discriminate between patients with

and without an abnormality in platelet function as detected by lumi-aggregometry, and showed

good levels of agreement with this reference test.

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All the participants identified by lumi-aggregometry to have a defect in dense granule

secretion or in the TxA2 pathway also presented with an abnormality on RPFT. Among the

participants with a defect in dense granule secretion, one had almost absent ATP release on lumi-

aggregometry and low CD63 expression on RPFT and was later identified to have Hermansky-

Pudlak syndrome-7 [12]. Interestingly, two other participants from the same group of defects

(highlighted in Figure 2b) presented with reduced P-selectin expression, but elevated CD63

expression even at baseline, and were later identified to have Hermansky-Pudlak syndrome-2,

which is known to be associated with impaired sorting of CD63 and its accumulation in the plasma

membrane rather than granule membrane [13].

Three individuals who were diagnosed with a defect in Gi signalling with lumi-aggregometry

appeared normal on RPFT. Their phenotype was relatively mild and the diagnosis was based

mainly on the kinetic information, with transient rather than sustained aggregation being

observed, or with delayed aggregation or secretion.

The RPFT identified 6 participants with reduced platelet reactivity that was not detected by

lumi-aggregometry. One of these participants had reduced P-selectin but normal CD63 expression

in response to all agonists, suggesting impaired α-granule secretion, which is reminiscent of Gray

Platelet Syndrome, a defect that would not be detected with lumi-aggregometry. These results

confirm that the absence of a platelet abnormality on traditional testing cannot eliminate the

possibility of a platelet defect.

There is considerable variability in methodology of LTA with a historical lack of

standardisation of aggregation [14]. Recently, the Platelet Physiology Scientific and

Standardization Committee of the International Society of Thrombosis and Haemostasis has

begun to standardise light transmission aggregometry [15] to improve the comparability of results

between laboratories. However, there remain many analytical and pre-analytical variables, which

render the application of lumi-aggregometry to a large population challenging. The main

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advantage of the RPFT is the use of whole blood. Furthermore, the analysis is performed on fixed

samples and thus can be done remotely without the need for immediate processing of the blood

sample. This offers the potential for standardisation and improved accessibility of platelet

function testing, which is currently restricted to specialised laboratories.

The performance of the RPFT in this study indicates that if it had been used as a pre-test,

the time and labour intensive lumi-aggregometry approach could have been avoided in over one

third of participants in whom no abnormality was detected using the traditional testing. Further

development of the RPFT should include the measurement of glycoprotein receptors and their

function, including GPVI, and platelet procoagulant function, which could improve the sensitivity

of this test and would add to the measurements that are lacking in traditionally available

methodologies [2]. These additions would require extra flow cytometry analyses, but would not

add complexity at the point-of-care.

We conclude that, while the RPFT would not be suitable for the comprehensive diagnosis of

PFDs, due to the limited number of agonists and absence of kinetic information, we believe it has

a role as a pre-test to identify subjects with a potential defect who would benefit from further

detailed platelet phenotyping. In subjects with a minimal history of excessive bleeding, it may also

help to identify those who are unlikely to have a defect in platelet function detectable by lumi-

aggregometry and thus do not require such testing. Further development of the RPFT and

research in the utility of this assay as a first-line screening test in a wider population of individuals

with bleeding symptoms is warranted.

Funding sources: Birmingham-Nottingham Strategic Collaboration Fund, British Heart Foundation

(RG/09/007/27917; PG/10/36/02) and Wellcome Trust (093994).

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Addendum

N. Dovlatova, M. Lordkipanidzé and G. C. Lowe contributed to the concept and design of the

study, collection, analysis and interpretation of data, critical writing and final approval of the

manuscript. B. Dawood and J. May contributed to the collection and analysis of data. S

Heptinstall, S. P. Watson and S. C. Fox contributed to the concept and design of the study, critical

writing and revising the intellectual content and final approval of the version of manuscript to be

published.

Acknowledgements

We would like to thank Dr Paul Harrison for his critical advice on the interpretation of the study

results and for constructive comments on the manuscript. We are also grateful to Milan Fernando

and Marko Benner for their technical support.

Legends

Figure 1. Receiver operating characteristic curves for P-selectin (a) and CD63 (b) expression

measurement with RPFT. Receiver operating characteristic curves demonstrating the relationship

between defects in platelet function as determined by lumi-aggregometry and a) P-selectin and b)

CD63 expression measurements with three conditions of platelet stimulation.

Table. Two-by-two analysis of the agreement between lumi-aggregometry and the RPFT.

«Positive» indicates the presence of an abnormality in platelet function and «negative» indicates

normal platelet responses. PPV and NPV stand for positive and negative predictive values

respectively.

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Figure 2. Performance of the RPFT in healthy subjects and in participants with platelet function

defects as determined by lumi-aggregometry. a) P-selectin and CD63 measured with RPFT in

healthy volunteers at baseline and after stimulation. For P-selectin cut-offs were 899 (ADP/U4),

991 (TRAP) and 1006 (AA/EPI), and for CD63 – 103 (ADP/U4), 120 (TRAP) and 112,5 (AA/EPI),

expressed as median fluorescence units; b) dense granule secretion defect (the two highlighted

participants are discussed in the text); c) defect in the TxA2 pathway and d) defect in Gi signalling.

Horizontal lines indicate cut-offs determined for each parameter using ROC curve analysis.

Figure 3. Platelet aggregation assessed by LTA in healthy volunteers (filled symbols) and in

patients with a defect in Gi signalling (open symbols). Platelet aggregation was assessed by LTA

in response to ADP (10 and 30μM, final aggregation) and epinephrine (10 and 30μM) in

participants with defect in Gi signalling. Horizontal lines indicate cut-offs determined as 5th

percentile of the corresponding measurement in healthy volunteers.

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Evaluation of a whole blood remote platelet function test for the diagnosis of mild bleeding disorders