Usefulness of Flow Cytometric Mepacrine Uptake/Release Combined with CD63 Assay in Diagnosis of Patients with Suspected Platelet Dense Granule Disorder

Abstract

Dense granule disorder is one of the most common platelet abnormalities, resulting from dense granule deficiency or secretion defect. This study was aimed to evaluate the clinical usefulness of the flow cytometric combination of mepacrine uptake/release assay and CD63 expression detection in the management of patients with suspected dense granule disorder. Over a period of 5 years, patients with abnormal platelet aggregation and/or reduced adenosine triphosphate (ATP) secretion suggestive of dense granule disorder were consecutively enrolled. The flow cytometric assays were systematically performed to further investigate dense granule functionality. Among the 26 included patients, 18 cases showed impaired mepacrine uptake/release and reduced CD63 expression on activated platelets, consistent with δ-storage pool deficiency (SPD). Another seven patients showed decrease in mepacrine release and CD63 expression but mepacrine uptake was normal, indicating secretion defect rather than δ-SPD. Unfortunately, ATP secretion could not be measured in 7 out of the 26 patients due to insufficient sample and/or severe thrombocytopenia. This test combination provides a rapid and effective method to detect the heterogeneous abnormalities of platelet dense granule by distinguishing between storage and release defects. This combination is particularly advantageous for severely thrombocytopenic patients and pediatric patients in which only minimal sample is required.

Keywords

dense granule disorder - mepacrine - CD63 - granulophysin - flow cytometry

• References

• 1 Reed GL. Chapter 15-Platelet secretion. In: Michelson A, ed. Platelets. London: Elsevier Science; 2007: 309-318
• 2 Mirlashari MR, Ryningen A, Mikkelsen HM, Fukami MH. Differential secretion of blood platelet storage granules. Platelets 1996; 7 (5–6) 313-320
• 3 Rivera J, Lozano ML, Navarro-Núñez L, Vicente V. Platelet receptors and signaling in the dynamics of thrombus formation. Haematologica 2009; 94 (5) 700-711
• 4 White JG. Use of the electron microscope for diagnosis of platelet disorders. Semin Thromb Hemost 1998; 24 (2) 163-168
• 5 Israels SJ, McMillan-Ward EM. Palmitoylation supports the association of tetraspanin CD63 with CD9 and integrin alphaIIbbeta3 in activated platelets. Thromb Res 2010; 125 (2) 152-158
• 6 McNicol A, Israels SJ. Platelet dense granules: structure, function and implications for haemostasis. Thromb Res 1999; 95 (1) 1-18
• 7 Nishibori M, Cham B, McNicol A, Shalev A, Jain N, Gerrard JM. The protein CD63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlak syndrome, and appears identical to granulophysin. J Clin Invest 1993; 91 (4) 1775-1782
• 8 Podda G, Femia EA, Pugliano M, Cattaneo M. Congenital defects of platelet function. Platelets 2012; 23 (7) 552-563
• 9 Hayward CP. Diagnostic evaluation of platelet function disorders. Blood Rev 2011; 25 (4) 169-173
• 10 Harrison P, Mackie I, Mumford A , et al; British Committee for Standards in Haematology. Guidelines for the laboratory investigation of heritable disorders of platelet function. Br J Haematol 2011; 155 (1) 30-44
• 11 Rao AK, Jalagadugula G, Sun L. Inherited defects in platelet signaling mechanisms. Semin Thromb Hemost 2004; 30 (5) 525-535
• 12 Mumford AD, Frelinger III AL, Gachet C , et al. A review of platelet secretion assays for the diagnosis of inherited platelet secretion disorders. Thromb Haemost 2015; 114 (1) 14-25
• 13 McNicol A, Israels SJ, Robertson C, Gerrard JM. The empty sack syndrome: a platelet storage pool deficiency associated with empty dense granules. Br J Haematol 1994; 86 (3) 574-582
• 14 Cattaneo M, Cerletti C, Harrison P , et al. 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 (6) 1183-1189
• 15 Cattaneo M. Light transmission aggregometry and ATP release for the diagnostic assessment of platelet function. Semin Thromb Hemost 2009; 35 (2) 158-167
• 16 Gordon N, Thom J, Cole C, Baker R. Rapid detection of hereditary and acquired platelet storage pool deficiency by flow cytometry. Br J Haematol 1995; 89 (1) 117-123
• 17 Rendu F, Nurden AT, Lebret M, Caen JP. Relationship between mepacrine-labelled dense body number, platelet capacity to accumulate 14C-5-HT and platelet density in the Bernard-Soulier and Hermansky-Pudlak syndromes. Thromb Haemost 1979; 42 (2) 694-704
• 18 Béri-Dexheimer M, Latger-Cannard V, Philippe C , et al. Clinical phenotype of germline RUNX1 haploinsufficiency: from point mutations to large genomic deletions. Eur J Hum Genet 2008; 16 (8) 1014-1018
• 19 Tosetto A, Castaman G, Rodeghiero F. Assessing bleeding in von Willebrand disease with bleeding score. Blood Rev 2007; 21 (2) 89-97
• 20 Tosetto A, Castaman G, Plug I, Rodeghiero F, Eikenboom J. Prospective evaluation of the clinical utility of quantitative bleeding severity assessment in patients referred for hemostatic evaluation. J Thromb Haemost 2011; 9 (6) 1143-1148
• 21 Bonduel M, Frontroth JP, Hepner M, Sciuccati G, Feliú-Torres A. Platelet aggregation and adenosine triphosphate release values in children and adults. J Thromb Haemost 2007; 5 (8) 1782-1783
• 22 Dawood BB, Wilde J, Watson SP. Reference curves for aggregation and ATP secretion to aid diagnose of platelet-based bleeding disorders: effect of inhibition of ADP and thromboxane A(2) pathways. Platelets 2007; 18 (5) 329-345
• 23 Wall JE, Buijs-Wilts M, Arnold JT , et al. A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents. Br J Haematol 1995; 89 (2) 380-385
• 24 Mullier F, Latger-Cannard V, Toussaint-Hacquard M , et al. Validation and assessment of mepacrine testing in delta storage pool disease: a 3-centre study. P-TH-089. J Thromb Haemost 2011; 9 (Suppl. 02) 797
• 25 Tosetto A, Castaman G, Rodeghiero F. Bleeders, bleeding rates, and bleeding score. J Thromb Haemost 2013; 11 (Suppl. 01) 142-150
• 26 Lowe GC, Lordkipanidzé M, Watson SP ; UK GAPP Study Group. Utility of the ISTH bleeding assessment tool in predicting platelet defects in participants with suspected inherited platelet function disorders. J Thromb Haemost 2013; 11 (9) 1663-1668
• 27 Hayward CP, Moffat KA. Laboratory testing for bleeding disorders: strategic uses of high and low-yield tests. Int J Lab Hematol 2013; 35 (3) 322-333
• 28 Hayward CP, Moffat KA, Pai M , et al. 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 (1) 134-145
• 29 Born GV, Wright HP. Platelet adhesiveness in experimental scurvy. Lancet 1967; 1 (7488) 477-478
• 30 Dey F, Möller A, Kemkes-Matthes B , et al. Reduced platelet aggregation in a boy with scurvy. Klin Padiatr 2012; 224 (7) 448-452
• 31 Weiss HJ, Lages B, Vicic W, Tsung LY, White JG. Heterogeneous abnormalities of platelet dense granule ultrastructure in 20 patients with congenital storage pool deficiency. Br J Haematol 1993; 83 (2) 282-295