Laboratory testing for platelet function disorders

Transcription

1 International Journal of Laboratory Hematology REVIEW The Official journal of the International Society for Laboratory Hematology INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY Laboratory testing for platelet function disorders S. J. ISRAELS Department of Pediatrics and Child Health, and Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada Correspondence: Dr Sara J. Israels, Department of Pediatrics and Child Health, University of Manitoba, 675 McDermot Ave., Winnipeg, Manitoba, Canada R3E 0V9. Tel.: ; Fax: ; doi: /ijlh Received 7 January 2015; accepted for publication 6 March 2015 Keywords Platelet disorders, platelet function tests, platelet aggregation, thrombocytopenia, clinical laboratory techniques/ standards SUMMARY Platelet function testing is both complex and labor intensive. A stepwise approach to the evaluation of patients with suspected platelet disorders will optimize the use of laboratory resources, beginning with an appropriate clinical evaluation to determine whether the bleeding is consistent with a defect of primary hemostasis. Bleeding assessment tools, evaluation of platelet counts, and review of peripheral blood cell morphology can aid the initial assessment. For patients requiring further laboratory testing, platelet aggregometry, secretion assays, and von Willebrand factor assays are the most useful next steps and will direct further specialized testing including flow cytometry, electron microscopy, and molecular diagnostics. Guidelines and recommendations for standardizing platelet function testing, with a particular focus on light transmission aggregometry, are available and can provide a template for clinical laboratories in establishing procedures that will optimize diagnosis and assure quality results. This review outlines an approach to platelet function testing and reviews testing methods available to clinical laboratories. INTRODUCTION The diagnostic evaluation of platelet disorders can be complex and time consuming. These disorders are diverse in their laboratory presentation, and therefore, developing a robust testing algorithm for the clinical laboratory can be a challenge. Until recently, there were no published recommendations regarding standardization of testing or interpretation, and it is even more recently that we have had access to external proficiency testing to aid quality assurance. Since 2008, four independent organizations have published recommendations or guidelines that address platelet function testing in general [1, 2] or light transmission aggregometry (LTA) in particular [3, 4]. The development of these guidelines was, in several cases, preceded by surveys of clinical and/or research laboratories engaged in platelet function testing that demonstrated marked variations in sample collection and preparation, assay parameters, and interpretation [5, 6]. This was particularly true for LTA, usually considered the essential laboratory platelet function assessment for the evaluation of clinical bleeding, 18

2 S. J. ISRAELS PLATELET FUNCTION TESTING 19 which was being applied with differences significant enough to impede comparison of results between laboratories. The purpose of this review is to highlight some key factors in clinical laboratory testing of platelet function that contribute to the quality of results and interpretation. PRIMARY HEMOSTASIS Circulating platelets monitor the integrity of the endothelial lining of blood vessels. When the blood vessel wall is injured and the endothelium is damaged, platelets adhere to the exposed subendothelial matrix proteins, initiating activation events that result in formation of a hemostatic plug [7]. Platelets adhere to collagen in the subendothelium directly, via their glycoprotein (GP) VI and integrin a2b1 membrane receptors, and to collagen-bound von Willebrand factor (VWF), at sites of higher shear (>1000 s 1 ) such as small arteries and the microvasculature, via GPIba of the GPIb-IX-V membrane receptor complex. Intracellular signaling events initiated by platelet adhesion result in reorganization of the platelet cytoskeleton leading to platelet shape change to irregular spheres with extended filopodia and spreading on the subendothelium; secretion of the contents of storage granules (e.g., ADP and serotonin from dense granules, and fibrinogen and growth factors from a-granules); formation of thromboxane A 2 (TxA 2 ) from arachidonic acid via cyclooxygenase-1 and thromboxane synthase; and procoagulant phosphatidylserine exposure on the platelet surface that accelerates thrombin generation. By binding to their specific membrane receptors, platelet agonists ADP, TxA 2, and thrombin initiate signaling pathways that convert integrin aiibb3 to a conformation with high affinity for divalent fibrinogen and, at high shear, multivalent VWF. These ligands function as bridges between aiibb3 on adjacent activated platelets, mediating aggregation. Hemostasis can be impaired if any of these processes in adhesion, activation, or aggregation are defective [8]. CLINICAL EVALUATION The assessment of patients should begin in the clinic with a detailed bleeding history, family history, and examination. This assessment can be aided by the use of one of several validated bleeding assessment tools (BATs), useful in standardizing information obtained from the patient history and accurately recording the severity and frequency of bleeding symptoms [9]. The high negative predictive value of some BATs may, in the future, make it possible to use them as a screen prior to laboratory testing. However, existing tools have low specificity and will not provide a definitive diagnosis [9, 10]. Mucocutaneous bleeding may be the result of inherited or acquired defects in platelet number and/or function or von Willebrand disease (VWD). In the absence of a specific family history, consideration of all of these possibilities must be entertained because bleeding symptoms alone will not differentiate among them. An algorithmic approach can be helpful in organizing the clinical and laboratory investigation of these patients, and several algorithms for the diagnosis of platelet disorders have been published [11 13]. Ideally, platelet function testing should be conducted in laboratories with specialized expertise and an adequate frequency/volume of testing to maintain technological proficiency. Laboratory results should be interpreted in the context of clinical information. LABORATORY TESTING Platelet count and blood film The initial laboratory investigation should include an automated complete blood count and peripheral blood film analysis, which are sensitive and specific for abnormalities of platelet number and can guide further laboratory investigations. Although providing accurate platelet counts within a broad range, standard automated impedance and optical platelet counting methods are less accurate when platelet counts are < /L or when platelets are significantly large. In these situations, immuno-counting using flow cytometric detection of fluorescent antibodylabeled platelets should be considered [14]. The mean platelet volume (MPV) is a value derived by most automated cell counters from measured platelet parameters and is influenced by blood sample collection and analysis [14]. Assuming that these are standardized, obtaining an accurate MPV can still be problematic for samples with macrothrombocytopenia. Evaluation of a Wright s or May-Gr unwald-giemsa-

3 20 S. J. ISRAELS PLATELET FUNCTION TESTING stained peripheral blood film provides additional information about platelet number, size, clumping, and granularity, as well as the morphology of erythrocytes and granulocytes. Pseudothrombocytopenia resulting from clumping of platelets collected in EDTA can also be identified and confirmed by re-collecting a specimen in a citrate-based anticoagulant. The presence of thrombocytopenia does not rule out platelet dysfunction; some inherited platelet disorders are characterized by both decreased platelet numbers and abnormal function [12, 15]. Global tests of hemostatic function and testing for VWD There is no ideal simple, inexpensive, and sensitive screening test that reliably identifies patients requiring specialized testing of platelet function. Although both bleeding times and PFA-100/200â closure times have been used for this purpose, these tests are not adequately sensitive to determine the need for further testing in patients with mucocutaneous bleeding, and their lack of specificity for platelet disorders limits their usefulness [16, 17]. Although they may have a role in the comprehensive evaluation of primary hemostatic abnormalities, these tests should be considered optional. Specific testing for VWD, either prior to, or concurrently with, platelet function testing is recommended. VWD should also be considered in patients with thrombocytopenia, as type 2B and platelet-type VWD can present with macrothrombocytopenia [18]. Platelet function testing Light Transmission Aggregometry The most widely used method of assessing platelet function is LTA, in which the change in optical density of a rapidly stirred sample of citrated platelet-rich plasma (PRP) at 37 C is measured by a photometer. Upon addition of agonists the platelets change shape from disks to rounded forms with extended filopodia, resulting in a transient, small decrease in light transmission that is followed by an increase as the platelets aggregate in a fibrinogen-dependent manner. Typically, the maximum increase in light transmission (% aggregation) is measured. The secondary wave of aggregation observed with higher concentrations of ADP and epinephrine is due to TxA 2 formation and secretion of granule contents. Platelet agglutination stimulated by ristocetin, which changes the conformation of plasma VWF allowing it to bind to GPIba, can also be measured by LTA. Standardization of LTA has recently been addressed in published guidelines and recommendations [3, 4], and although some specific recommendations differ among these guides, there are many areas of consensus that make LTA best practices for individual clinical laboratories an achievable goal. Pre-analytical Variables. Light transmission aggregometry is significantly impacted by differences in sample collection and preparation. Standardizing preanalytical and analytical variables can improve the consistency of results. Ideally, samples should be collected from patients who have refrained from smoking or caffeine intake before the collection and after a short period of rest [2, 4]. A detailed medication history for the preceding 1 2 weeks should be obtained at the time of collection. Patients who are able to stop medications that affect platelet function should be instructed to do so for 7 10 days before the date of testing [2, 4]. However, many patients cannot/should not discontinue their medications, and therefore, a record of medications should accompany the specimen to the laboratory to aid the interpretation of the results [2 4]. This record should include food and herbs that may impact platelet function, in addition to medications [2]. Blood should be collected using atraumatic venipuncture with minimal tourniquet pressure into buffered citrate anticoagulant to maintain a stable ph until testing, and the first few ml of blood drawn should be discarded or used for other tests. PRP should be prepared from whole blood by centrifuging samples at 200 g for 10 min at ambient temperature without application of a brake [1, 2, 4]. The samples should be examined for the evidence of gross hemolysis and lipemia, both of which will affect light transmission [1, 2, 4]. The platelet concentration of the PRP should be measured. LTA studies of PRP with a count of < /L should be interpreted with caution (Hayward et al. [19] provide a guide for evaluation of thrombocytopenic samples). Some guidelines recommend against adjustment of the PRP platelet count to a standardized value if it is < /L [2, 4]. Studies have demonstrated no advantage to standardizing

4 S. J. ISRAELS PLATELET FUNCTION TESTING 21 the platelet count, and the additional manipulation may affect in vitro platelet responses [20, 21]. The PRP should be allowed to rest at room temperature for 15 min before testing, and testing should be completed within 4 h of blood collection [1, 2, 4]. Analytical Variables. Clinical laboratories should develop a standard operating procedure for performing LTA that includes choice of a standard panel of agonists and agonist concentrations. Each laboratory must establish reference intervals for each agonist (and concentration) and validate test performance with each new lot of reagent [1, 3]. Ideally, normal cont-rol samples will be run in parallel with patient samples [2 4]. Before adding an agonist, a baseline tracing should be observed for stability and oscillations. Following addition of the agonist, the aggregation response should be monitored for a minimum of 3 5 min, and longer if the agonist does not produce maximal aggregation in normal controls by that time [2]. An exhaustive menu of agonists is not required for initial testing of patients [22, 23], but should include a standard panel with agonist concentrations chosen to optimize the identification of abnormal responses. A suggested basic panel is shown in Table 1 and includes single concentrations of ADP, collagen, epinephrine, arachidonic acid, and ristocetin [1 4]. An extended agonist panel can be used as reflexive testing when results from the basic panel are abnormal. If the initial aggregation response to an agonist is abnormal, then retesting with higher concentrations should be performed. If response to 1.2 mg/ml of ristocetin is normal, then a low concentration of ristocetin ( mg/ml) should be used to check for an increased agglutination response associated with type 2B or platelet-type VWD. If response to arachidonic acid is decreased, using the thromboxane mimetic U46619 will differentiate an aspirin-like effect from thromboxane receptor abnormalities. Extended panels sometimes include agonists such as thrombin receptor activation peptides (TRAPs), collagen-related peptide or convulxin, and calcium ionophore A23187, which can provide additional diagnostic information in specific cases [2]. The aggregation tracings should be examined for the quality of the baseline, the presence of shape change, the length of the lag phase, the slope of the aggregation curve, the maximum percent aggregation, and the presence of deaggregation. Examples of LTA patterns associated with selected platelet abnormalities are shown in Table 2. Whole Blood Aggregometry Whole blood aggregometry (WBA) measures aggregation as the change in electrical impedance between two electrodes as platelets adhere and aggregate in Table 1. Suggested agonist panel for light transmission aggregometry* Agonist Starting concentration Concentration range Target Basic Panel ADP lm lm P2Y 1 and P2Y 12 Epinephrine 5 lm lm Adrenergic receptors Collagen (type I) 1 2 lg/ml lg/ml GPVI and a2b1 Arachidonic Acid 1 mm mm TxA 2 synthesis Ristocetin 1.2 mg/ml 0.5 mg/ml mg/ml mg/ml VWF/GPIb Extended Panel U lm 1 5 lm TxA 2 receptor TRAP 10 lm lm PAR-1 CRP 10 ng/ml ng/ml GPVI A lm 1 10 lm Calcium mobilization TxA2, thromboxane A 2 ; VWF, von Willebrand factor; TRAP, thrombin receptor activating peptide; CRP, collagenrelated peptide; PAR-1, protease-activated receptor-1. *Adapted with modifications from References [2] and [4].

5 22 S. J. ISRAELS PLATELET FUNCTION TESTING Table 2. Light transmission aggregation responses for some platelet function disorders Disorder LTA responses Additional observations or testing Bernard-Soulier syndrome Type 2B VWD and platelet-type VWD Glanzmann thrombasthenia Aspirin-like defect Secretion defect and d-granule defect ADP receptor defect Gray platelet syndrome Absent agglutination response to ristocetin Increased agglutination with low concentrations of ristocetin Absent response to all agonists except ristocetin Absent response to arachidonic acid with normal response to U46619; decreased responses to ADP and low concentrations of collagen [second wave] May show decreased response to several agonists: ADP, collagen and epinephrine Decreased or absent response to ADP May show decreased response to thrombin and/or collagen Macrothrombocytopenia Rule out VWD Flow cytometry for GPIb quantitation Macrothrombocytopenia and platelet clumping may be present. VWD testing required Flow cytometry for aiibb3 quantitation Medication history for COX-1 inhibitors ATP release and/or electron microscopy for d-granule evaluation Medication history for ADP receptor inhibitors Flow cytometry for P2Y 12 quantitation Macrothrombocytopenia with pale platelets on blood film LTA, light transmission aggregometry; VWD, von Willebrand disease; COX, cyclooxygenase. response to agonists. Adherent platelets increase the impedance, which is displayed as an aggregation curve. WBA has the advantages of requiring smaller blood volumes and less manipulation of the sample than LTA [1]. Fully automated aggregometers with disposable electrodes are available making it a feasible alternative to LTA (Multiplate â analyzer), although not the choice of most clinical laboratories at present [2]. Agonist responsiveness differs between LTA and WBA, and direct comparison studies of the two methodologies are still required [24, 25]. Secretion assays: ATP release The measurement of released nucleotides from platelet dense granules is usually performed as an adjunct to aggregation studies, and if decreased indicates abnormalities of platelet dense granule number, content, or secretion. LTA alone is not adequately sensitive to identify all patients with secretion defects, which are likely to be under-diagnosed without a measurement of dense granule secretion [26]. The most frequently used assay measures agonist stimulated release of dense granule ATP using a lumi-aggregometer, which detects light emitted from ATP reacting with the bioluminescent reagent luciferin/luciferase [26, 27]. This assay requires that samples be normalized to a standardized platelet count in order to make results comparable to a laboratory-established ATP standard and does not differentiate between abnormalities of dense granule per se and disorders of secretion. Quantitation of platelet dense granules by whole mount electron microscopy will identify patients with decreased ATP release secondary to decreased numbers of dense granules [28]. Alternative methods for the measurement of total nucleotide content, and release can be performed by luminometry or high performance liquid chromatography. Uptake and release of mepacrine or radiola-

6 S. J. ISRAELS PLATELET FUNCTION TESTING 23 beled serotonin can also be used to evaluate dense granule secretion [1, 29]. Flow cytometry For specific patients with a clinical history and/or platelet function studies that suggest a diagnosis of Bernard-Soulier syndrome or Glanzmann thrombasthenia, flow cytometry to quantitate the appropriate membrane glycoprotein receptor density (GPIb-IX-V, aiibb3) can be used to investigate and confirm these diagnoses. Flow cytometry can also be employed to identify quantitative abnormalities of receptors for collagen (GPVI, a2b1) or thrombin (PAR-1) and to measure platelet agonist-induced responses including the exposure of anionic phospholipids [29]. Genotyping Molecular genetic testing can provide confirmation of the diagnosis for heritable platelet disorders (thrombocytopenias and function defects) where the genetic basis is known. As new causative genes are identified, the application of molecular diagnostics will increase, particularly when phenotypic studies are not feasible, and for antenatal diagnosis [15]. Genetic loci associated with platelet disorders are detailed in several recent reviews [15, 30]. Other assays There are a variety of additional instruments/assays for point-of-care and near point-of-care testing of platelet function [29]. The major focus of many of these instruments has been the monitoring of antiplatelet therapy in the setting of cardiovascular disease (e.g., VerifyNowâ, Impact-R TM, Plateletworksâ) orin the surgical setting (thromboelastography). Most are either not suited for, or have not been adequately validated for, the diagnosis of platelet function disorders. An exception is the Optimul 96-well microtiter plate aggregation assay, which allows high throughput assessment of platelet aggregation responses to multiple agonists simultaneously, and has shown reasonable concordance with LTA [31]. Newer technologies, including microfluidics, hold the promise of changing how we study in vitro platelet function in the future. QUALITY ASSURANCE Although external proficiency testing (EPT) has been part of a quality assurance program for hemostasis laboratories for many years, platelet function testing has presented unique challenges for EPT programs, because of the complexities of sample preparation and shipping. However, a number of organizations now offer EPT for the evaluation of closure times, LTA, dense granule counting by whole mount EM, and clinical case interpretation [32]. SUMMARY A pragmatic stepwise approach is required for platelet function testing. Clinical evaluation should be the first step in determining the need for laboratory testing, and the use of a diagnostic algorithm can rationalize the investigation strategy for clinicians and laboratories. In most cases, the first level of testing should include platelet count, blood film, LTA with a standard panel of agonists, and secretion assay or other measure of platelet nucleotide content. More specialized testing can be reserved for patients whose initial investigations (both clinical and laboratory-based) suggest platelet function abnormalities. These additional investigations may include an expanded LTA panel, electron microscopy, flow cytometry, and/or molecular genetic testing. CONFLICT OF INTEREST The authors declare no conflict of interest. REFERENCES 1. Christie DJ, Avari T, Carrington LR, Cohen E, DeBiase BA, Harrison P, Kickler TS, Kottke-Marchant K, Ledford-Kraemer M, Rand ML, Schmaier AH, McCabe White M. Plate-let Function Testing by Aggregometry; Approved Guideline. Wayne, PA: Clinical and Laboratory Standards Institute; 2008; 28: Harrison P, Mackie I, Mumford A, Briggs C, Liesner R, Winter M, Machin S, British

7 24 S. J. ISRAELS PLATELET FUNCTION TESTING Committee for Standards in Haematology. Guidelines for the laboratory investigation of heritable disorders of platelet function. Br J Haematol 2011;155: Hayward CP, Moffat KA, Raby A, Israels S, Plumhoff E, Flynn G, Zehnder JL. Development of North American consensus guidelines for medical laboratories that perform and interpret platelet function testing using light transmission aggregometry. Am J Clin Pathol 2010;134: Cattaneo M, Cerletti C, Harrison P, Hayward CP, Kenny D, Nugent D, Nurden P, Rao AK, Schmaier AH, Watson SP, Lussana F, Pugliano MT, Michelson AD. Recommendations for the standardization of light transmission aggregometry: a consensus of the working party from the platelet physiology subcommittee of SSC/ISTH. J Thromb Haemost 2013;11: Cattaneo M, Hayward CP, Moffat KA, Pugliano MT, Liu Y, Michelson AD. Results of a worldwide survey on the assessment of platelet function by light transmission aggregometry: a report from the platelet physiology subcommittee of the SSC of the ISTH. J Thromb Haemost 2009;7: Moffat KA, Ledford-Kraemer MR, Nichols WL, Hayward CP. North American Specialized Coagulation Laboratory A. Variability in clinical laboratory practice in testing for disorders of platelet function: results of two surveys of the North American Specialized Coagulation Laboratory Association. Thromb Haemost 2005;93: Ruggeri ZM, Jackson SP. Platelet thrombus formation in flowing blood. In: Platelets, 3rd edn. Michelson AD (ed.). London: Academic Press, 2013: Israels SJ, Rand ML. What we have learned from inherited platelet disorders. Pediatr Blood Cancer 2013;60(Suppl 1):S Rydz N, James PD. The Evolution and value of bleeding assessment tools. J Thromb Haemost 2012;10: Lowe GC, Lordkipanidze M, Watson SP. Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders. J Thromb Haemost 2013;11: Favaloro EJ, Lippi G, Franchini M. Contemporary platelet function testing. Clin Chem Lab Med 2010;48: Israels SJ, Kahr WH, Blanchette VS, Luban NL, Rivard GE, Rand ML. Platelet disorders in children: a diagnostic approach. Pediatr Blood Cancer 2011;56: Lambert MP. What to do when you suspect an inherited platelet disorder. Hematology Am Soc Hematol Educ Program 2011;2011: Briggs C, Harrison P, Machin SJ. Continuing developments with the automated platelet count. Int J Lab Hematol 2007;29: Nurden AT, Nurden P. Congenital platelet disorders and understanding of platelet function. Br J Haematol 2014;165: Cattaneo M. Are the bleeding time and PFA-100 useful in the initial screening of patients with mucocutaneous bleedings of hereditary nature? J Thromb Haemost 2004;2: Quiroga T, Goycoolea M, Munoz B, Morales M, Aranda E, Panes O, Pereira J, Mezzano D. Template bleeding time and PFA-100 have low sensitivity to screen patients with hereditary mucocutaneous hemorrhages: comparative study in 148 patients. J Thromb Haemost 2004;2: Jackson SC, Sinclair GD, Cloutier S, Duan Z, Rand ML, Poon MC. The Montreal platelet syndrome kindred has type 2B von Willebrand disease with the VWF V1316M mutation. Blood 2009;113: Hayward CP, Moffat KA, Pai M, Liu Y, Seecharan J, McKay H, Webert KE, Cook RJ, Heddle NM. An evaluation of methods for determining reference intervals for light transmission platelet aggregation tests on samples with normal or reduced platelet counts. Thromb Haemost 2008; 100: Cattaneo M, Lecchi A, Zighetti ML, Lussana F. Platelet aggregation studies: autologous platelet-poor plasma inhibits platelet aggregation when added to platelet-rich plasma to normalize platelet count. Haematologica 2007;92: Castilloux JF, Moffat KA, Liu Y, Seecharan J, Pai M, Hayward CP. A prospective cohort study of light transmission platelet aggregometry for bleeding disorders: is testing native platelet-rich plasma non-inferior to testing platelet count adjusted samples? Thromb Haemost 2011;106: Hayward CP, Pai M, Liu Y, Moffat KA, Seecharan J, Webert KE, Cook RJ, Heddle NM. Diagnostic utility of light transmission platelet aggregometry: results from a prospective study of individuals referred for bleeding disorder assessments. J Thromb Haemost 2009;7: Dawood BB, Lowe GC, Lordkipanidze M, Bem D, Daly ME, Makris M, Mumford A, Wilde JT, Watson SP. Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel. Blood 2012;120: McGlasson DL, Fritsma GA. Whole blood platelet aggregometry and platelet function testing. Semin Thromb Hemost 2009;35: Peerschke EI, Castellone DD, Stroobants AK, Francis J. Reference range determination for whole-blood platelet aggregation using the multiplate analyzer. Am J Clin Pathol 2014;142: Pai M, Wang G, Moffat KA, Liu Y, Seecharan J, Webert K, Heddle N, Hayward C. Diagnostic usefulness of a lumi-aggregometer adenosine triphosphate release assay for the assessment of platelet function disorders. Am J Clin Pathol 2011;136: Cattaneo M. Light transmission aggregometry and ATP release for the diagnostic assessment of platelet function. Semin Thromb Hemost 2009;35: White JG. Electron microscopy methods for studying platelet structure and function. Methods Mol Biol 2004;272: Harrison P, Lordkipanidze M. Testing platelet function. Hematol Oncol Clin North Am 2013;27: Bunimov N, Fuller N, Hayward CP. Genetic loci associated with platelet traits and platelet disorders. Semin Thromb Hemost 2013;39: Lordkipanidze M, Lowe GC, Kirkby NS, Chan MV, Lundberg MH, Morgan NV, Bem D, Nisar SP, Leo VC, Jones ML, Mundell SJ, Daly ME, Mumford AD, Warner TD, Watson SP, UK Genotyping and Phenotyping of Platelets Study Group. Characterization of multiple platelet activation pathways in patients with bleeding as a high-throughput screening option: use of 96-well Optimul assay. Blood 2014;123: e Hayward CP, Moffat KA, Plumhoff E, Timleck M, Hoffman S, Spitzer E, Van Cott EM, Meijer P. External quality assessment of platelet disorder investigations: results of international surveys on diagnostic tests for dense granule deficiency and platelet aggregometry interpretation. Semin Thromb Hemost 2012;38: