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

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1 Journal of Thrombosis and Haemostasis, 12: DOI: /jth BRIEF REPORT Evaluation of a whole blood remote platelet function test for the diagnosis of mild bleeding disorders N. DOVLATOVA,* M. LORDKIPANIDZ E, G. C. LOWE, B. DAWOOD, J. MAY,* S. HEPTINSTALL,* S. P. WATSON and S. C. FOX,* FOR THE UK GAPP STUDY GROUP *Cardiovascular Medicine, University of Nottingham, Queens Medical Centre, Nottingham; and Centre for Cardiovascular Sciences, Birmingham Platelet Group, University of Birmingham, Birmingham, UK To cite this article: Dovlatova N, Lordkipanidze M, Lowe GC, Dawood B, May J, Heptinstall S, Watson SP, Fox SC, for the UK GAPP Study Group. Evaluation of a whole blood remote platelet function test for the diagnosis of mild bleeding disorders. J Thromb Haemost 214; 12: Summary. 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: A remote platelet function test was compared with lumi-aggregometry in participants recruited to the Genotyping and Phenotyping of Platelets Study (GAPP, ISRCTN ). For the 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: For the 61 study participants (42 index cases and 19 relatives) there was a good agreement between lumi-aggregometry and the RPFT, with diagnosis being concordant in 84% of cases (j =.668, P <.1). According to both tests, 29 participants were identified to have a deficiency in platelet function and 22 participants appeared normal. There were four participants where lumi-aggregometry revealed a defect but the RPFT did not, and six participants where the RPFT detected an abnormal platelet response that was not identified by lumi-aggregometry. Conclusion: This study suggests that the RPFT could be an easy-to-use pretest to select which participants with bleeding disorders Correspondence: Natalia Dovlatova, Cardiovascular Medicine, D Floor, South Block, Queen s Medical Centre, Nottingham NG7 2UH, UK. Tel.: ; fax: natalia.dovlatova@nottingham.ac.uk Received 8 November 213 Manuscript handled by: C. Gachet Final decision: F. R. Rosendaal, 23 February 214 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; flow cytometry; platelet function tests; platelets. Introduction Mild platelet function disorders (PFDs) are complex, poorly understood, difficult to diagnose and require extensive laboratory testing limited to specialized laboratories [1,2]. Lumi-aggregometry, where dense granule secretion is assessed in parallel with traditional LTA [3 5], is informative but is time and labor 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 6% of participants with excessive clinical bleeding and a suspected underlying inherited PFD have a demonstrable abnormality in platelet responses [5]. This suggests that 4% of patients may have a platelet abnormality that is not detectable by lumi-aggregometry or may have a defect in other components of the hemostatic 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. [6] where approximately 6% of a slightly different patient population with mild bleeding had no identified defect when assessed by LTA and serotonin and ATP release measurements. 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 characterization. At present, there is no reliable, easy-to-use and sufficiently sensitive global platelet function test available for use as a screening tool in subjects with suspected PFD [7]. 214 International Society on Thrombosis and Haemostasis

2 Remote platelet test for bleeding disorders 661 We have developed a remote platelet function test (RPFT), in which a blood sample is manipulated in a simple way that does not require specialized staff or equipment at the test site. The sample is stabilized utilizing a fixing solution, PAMFix, and sent to a flow cytometry facility for analysis [8], where the expression of the a-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 a-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 with lumiaggregometry in participants recruited to the Genotyping and Phenotyping of Platelets Study (GAPP, ISRCTN ) from April 212 to March 213. We studied 61 participants (42 index cases and 19 recruited relatives), 13 male (median age 13, range 5 45) and 48 female (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 [1]. 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 centers (maximum distance 1 miles) to the central laboratory as previously described [5]. This study was approved by the National Research Ethics Service Committee West Midlands Edgbaston (ref 6/MRE7/36). All participants provided written informed consent. Assessment of platelet function For the RPFT measurements, whole blood samples collected into sodium citrate were pre-warmed and stimulated at 37 C for 5 min without mixing. They were then fixed using PAMFix (Platelet Solutions Ltd, Nottingham, UK). Samples were then transported from the central laboratory to the flow cytometry facility (6 miles) and routinely analyzed within 3 days. For platelet stimulation, blood samples were treated with (i) saline alone to provide a baseline test, (ii) adenosine diphosphate (ADP) with U46619, a stable thromboxane A 2 analogue ( 1 and 1 lm, respectively), (iii) thrombin receptor activating peptide ( [SFLLRN] 2 lm) and (iv) arachidonic acid with epinephrine (,.5 mm and 1 lm, 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 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 twoby-two analysis and Cohen s kappa statistic. Analyses were performed using the software packages SPSS 14. (SPSS Inc, Chicago, IL, USA) and GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Results According to lumi-aggregometry, 33 of the 61 participants recruited to the 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 lumi-aggregation profile [5]: defects in dense granule secretion (n = 9), G i signalling (n = 14) and the TxA 2 pathway (n = 3). The remaining seven 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 (n = 2). The area under the ROC curve for P-selectin (Fig. 1A) and CD63 measurements (Fig. 1B) with different agonists varied between.72 and.99 (P <.4). This confirmed that the RPFT can discriminate between individuals with excessive bleeding with and without abnormal platelet 214 International Society on Thrombosis and Haemostasis

3 662 N. Dovlatova et al A P-selectin AUC =.75 P < AUC =.72 P < AUC =.85 P < Specificity (%) Specificity (%) Specificity (%) 1 B CD AUC =.89 P = AUC =.87 P = AUC =.99 P < Specificity (%) Specificity (%) Specificity (%) 1 Fig. 1. Receiver operating characteristic curves for P-selectin (A) and CD63 (B) expression measurement with the 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. 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 minimize false negative results. Applying the chosen cut-offs in the group of healthy controls indicated that five out of 41 participants presented with somewhat reduced platelet responses on RPFT (Fig. 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 hemostasis [6]. In the patient group, the overall agreement between lumi-aggregometry and the RPFT was good, with the diagnosis being concordant in 84% of cases (j =.668, P <.1) (Table 1). All participants who presented with a dense granule secretion defect (Fig. 2B) or a defect in the TxA 2 pathway (Fig. 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 G i signaling based on lumi-aggregometry (Fig. 2D), 11 also showed abnormal platelet responses on RPFT, while three subjects were considered normal. It should be noted that presentation of the G i -type platelet defect on lumi-aggregometry is variable, overlaps considerably with corresponding measurements in healthy volunteers (Fig. 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 a-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 214 International Society on Thrombosis and Haemostasis

4 Remote platelet test for bleeding disorders 663 A Performance of the RPFT in healthy subjects (n = 41) C RPFT in patients with a defect in TxA 2 pathway (n = 3) B RPFT in patients with a dense granule secretion defect (n = 9) 2 5 D RPFT in patients with a defect in G i signalling (n = 14) Fig. 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 () and 16 (), and for CD63 they were 13 (), 12 () and (), expressed as median fluorescence units. (B) Dense granule secretion defect (the two highlighted participants are discussed in the text). (C) Defect in the TxA 2 pathway and (D) defect in G i signaling. Horizontal lines indicate cut-offs determined for each parameter using ROC curve analysis Table 1 Two-by-two analysis of the agreement between lumi-aggregometry and the remote platelet function test (RPFT) RPFT positive RPFT negative Lumi-aggregometry positive Lumi-aggregometry negative 29 6 PPV = 83% 4 22 NPV = 85% Sensitivity = 88% Specificity = 79% 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. an inherited PFD. The new test directly assesses both a-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. All the participants identified by lumi-aggregometry to have a defect in dense granule secretion or in the TxA 2 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 lumiaggregometry and low CD63 expression on RPFT and was later identified to have Hermansky-Pudlak syndrome- 7 [12]. Interestingly, two other participants with a defect in dense granule secretion (highlighted in Fig. 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 the granule membrane [13]. Three individuals who were diagnosed with a defect in G i signaling 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 delayed aggregation or secretion. The RPFT identified six 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 a-granule secretion, which is reminiscent 214 International Society on Thrombosis and Haemostasis

5 664 N. Dovlatova et al % aggregation µm ADP final 31 µm ADP final 1 µm Epinephrine 3 µm Epinephrine Fig. 3. Platelet aggregation assessed by LTA in healthy volunteers (filled symbols) and in patients with a defect in G i signaling (open symbols). Platelet aggregation was assessed by LTA in response to ADP (1 and 3 lm, final aggregation) and epinephrine (1 and 3 lm) in participants with a defect in z i signaling. Horizontal lines indicate cut-offs determined as the 5th percentile of the corresponding measurement in healthy volunteers. 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 the methodology of LTA, with a historical lack of standardization of aggregation [14]. Recently, the Platelet Physiology Scientific and Standardization Committee of the International Society of Thrombosis and Haemostasis has begun to standardize 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 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 standardization and improved accessibility of platelet function testing, which is currently restricted to specialized laboratories. The performance of the RPFT in this study indicates that if it had been used as a pre-test, the time and labor 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 on the utility of this assay as a firstline screening test in a wider population of individuals with bleeding symptoms are warranted. Addendum N. Dovlatova, M. Lordkipanidze 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 of the intellectual content and final approval of the version of the manuscript to be published. Acknowledgements We would like to thank P. Harrison for his critical advice on the interpretation of the study results and for constructive comments on the manuscript. We are also grateful to M. Fernando and M. Benner for their technical support. Funding sources Birmingham-Nottingham Strategic Collaboration Fund, British Heart Foundation (RG/9/7/27917; PG/1/36/ 2) and Wellcome Trust (93994). Disclosure of Conflict of Interests The authors state that they have no conflict of interests. References 1 Watson SP, Lowe GC, Lordkipanidze M, Morgan NV; on behalf of the GAPP consortium. Genotyping and phenotyping of platelet function disorders. J Thromb Haemost 213; 11 (Suppl. 1): Quiroga T, Mezzano D. Is my patient a bleeder? A diagnostic framework for mild bleeding disorders. Hematology Am Soc Hematol Educ Program 212; 212: Miller JL. Platelet function testing: an improved approach utilizing lumi-aggregation and an interactive computer system. Am J Clin Pathol 1984; 81: Nieuwenhuis HK, Akkerman JW, Sixma JJ. Patients with a prolonged bleeding time and normal aggregation tests may have 214 International Society on Thrombosis and Haemostasis

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