European Working Group on Clinical Cell Analysis: Consensus Protocol for the Flow Cytometric Characterisation of Platelet Function

Introduction

An increased or disturbed activation and aggregation of platelets plays a major role in the pathophysiology of thrombosis and haemostasis and is related to cardiovascular disease processes. In addition to qualitative disturbances of platelet function, changes in thrombopoiesis or an increased elimination of platelets, (e. g., in autoimmune thrombocytopenia), are also of major clinical relevance. Flow cytometry is increasingly used for the specific characterisation of phenotypic alterations of platelets which are related to cellular activation, haemostatic function and to maturation of precursor cells. These new techniques also allow the study of the in vitro response of platelets to stimuli and the modification thereof under platelet-targeted therapy as well as the characterisation of platelet-specific antibodies. In this protocol, specific flow cytometric techniques for platelet analysis are recommended based on a description of the current state of flow cytometric methodology. These recommendations are an attempt to promote the use of these new techniques which are at present broadly evaluated for diagnostic purposes. Furthermore, the definition of the still open questions primarily related to the technical details of the method should help to promote the multi-center evaluation of procedures with the goal to finally develop standardized operation procedures as the basis of interlaboratory reproducibility when applied to diagnostic testing.

• References

• 1 Haslam RJ. Signal transduction in platelet activation in thrombosis and haemostasis. Verstrate M, Vermylen J, Lijnen HR, Arnout J. (eds.) 1987. p 147-74.
• 2 Abrams CS, Shattil SJ. Immunological detection of activated platelets in clinical disorders. Thromb Haemost 1991; 65: 467-73.
• 3 Janes SL, Wilson DJ, Chronos N, Goodall AH. Evaluation of whole blood flow cytometric detection of platelet bound fibrinogen on normal subjects and patients with activated platelets. Thromb Haemost 1993; 70: 659-66.
• 4 Michelson AD, Shattil SJ. The use of flow cytometry to study platelet activation. In: Watson SP, Authi KS. (eds.) Platelets. A Practical Approach. Oxford University Press; Oxford: 1996. pp 111-29.
• 5 Dachary-Prigent J, Pasquet JM, Freyssinet JM, Nurden AT. Calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: a study using Ca²⁺-ATPase inhibitors. Biochemistry 1995; 34: 11625-34.
• 6 Davies TA, Drotts D, Wie GJ, Simons ER. Flow cytometric measurement of cytoplasmic calcium changes in human platelets. Cytometry 1988; 9: 138-42.
• 7 Oda A, Daley JF, Cabral C, Kang J, Smith M, Salzman EW. Heterogeneity in filament actin content among individual human blood platelets. Blood 1992; 79: 920-7.
• 8 Ruf A, Patscheke H. Flow cytometric detection of activated platelets: comparison of determining shape change, fibrinogen binding, and P-selectin expression. Semin Thromb Hemostasis 1995; 21: 146-51.
• 9 Hourdille P, Heilmann E, Combrie R, Winckler J, Clementson KJ, Nurden AT. Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets. Blood 1990; 76: 1503-13.
• 10 Hourdille P, Gralnick HR, Heilmann E, Derlon A, Ferrer AM, Vezon G, Nurden AT. Von Willebrand factor bound to glycoprotein Ib is cleared from the platelet surface after platelet activation by thrombin. Blood 1992; 79: 2011-21.
• 11 Michelson AD, Benoit SE, Kroll MH, Li JM, Rohrer MJ, Kestin AS, Barnard MR. The activation-induced decrease in the platelet surface expression of the glycoprotein Ib-IX complex is reversible. Blood 1994; 83: 3562-73.
• 12 Rao GH, Peller JD, White JG. Influence of ionized calcium on thrombin-induced down regulation of GPIb/IX receptors on human platelets. Thromb Res 1997; 85: 23-31.
• 13 Wencel-Drake JD, Plow EF, Kunicki TJ, Woods VL, Keller DM, Ginsberg MH. Localization of internal pools of membrane glycoproteins involved in platelet adhesive responses. Am J Pathol 1986; 124: 324-34.
• 14 Woods Jr. VL, Wolff LE, Keller DM. Resting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins. J Biol Chem 1986; 261: 15242-51.
• 15 Sims PJ, Fianoni EM, Weidmer T, Shattil SJ. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and expression of prothrombinase activity. J Biol Chem 1988; 263: 18205-12.
• 16 Gilbert GE, Sims PJ, Wiedmer T, Furie B, Furie BC, Shattil SJ. Platelet-derived microparticles express high affinity receptors for factor VIII. J Biol Chem 1991; 266: 17261-8.
• 17 Hoffman M, Monroe DM, Roberts HR. Coagulation factor IXa binding to activated platelets and platelet-derived microparticles: a flow cytometric study. Thromb Haemost 1992; 68: 74-8.
• 18 Dachary-Prigent J, Freyssinet JM, Pasquet JM, Carron JC, Nurden AT. Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups. Blood 1993; 81: 2554-65.
• 19 Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70: 307-15.
• 20 Sims PJ, Ginsberg MH, Plow EF, Shattil SJ. Effect of platelet activation on the conformation of the plasma membrane glycoprotein IIb-IIIa complex. J Biol Chem 1991; 266: 7345-52.
• 21 Nurden P, Savi P, Heilmann E, Bihour C, Herbert J-M, Maffrand J-P, Nurden A. An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. J Clin Invest 1995; 95: 1612-22.
• 22 Frojmovic MM. Flow cytometric analysis of platelet activation and fibrinogen binding. Platelets 1996; 7: 9-21.
• 23 Ginsberg MH, Frelinger AL, Lam SCT, Forsyth J, McMillan R, Plow EF, Shattil SJ. Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies. Blood 1990; 76: 2017-23.
• 24 Ugarova TP, Budzynski AZ, Shattil SJ, Ruggeri ZM, Ginsberg MH, Plow EF. Conformational changes in fibrinogen elicited by its interaction with platelet membrane glycoprotein GpIIb/IIIa. J Biol Chem 1993; 268: 21080-7.
• 25 Metzelaar MJ, Clevers HC. Lysosomal membrane glycoproteins in platelets. Thromb Haemost 1992; 68: 378-82.
• 26 Febbraio M, Silverstein RL. Identification and characterisation of LAMP-1 as an activation-dependent platelet surface glycoprotein. J Biol Chem 1990; 256: 18531-7.
• 27 Silverstein RL, Febbraio M. Identification of lysosome-associated membrane protein-2 as an activation-dependent platelet surface glycoprotein. Blood 1992; 80: 1470-5.
• 28 Gerrard JM, Limt D, Sims PJ, Wiedmer T, Fugate D, McMillan E, Roberts C, Israels SJ. Identification of a platelet dense granule membrane protein that is deficient in a patient with Hermansky-Pudlak syndrome. Blood 1991; 77: 101-12.
• 29 McNicol A, Israels SJ. The lysosomal granule membrane protein, LAMP-2, is present in platelet dense granule membranes. Blood 1994; 84: 473a.
• 30 Israels SJ, Gerrard JM, Jacques YV, McNicol A, Cham B, Nishibori M, Bainton DF. Platelet dense granule membranes contain both granulophysin and P-selectin (GMP-140). Blood 1992; 80: 143-52.
• 31 Sternberg PE, McEver RP, Shuman MA, Jacques YV, Bainton DF. A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation. J Cell Biol 1985; 101: 880-6.
• 32 Metzelaar MJ, Heijnen HFG, Sixma JJ, Nieuwenhuis HK. Identification of a 33Kd protein associated with the a-granule membrane (GMP-33) that is expressed on the surface of activated platelets. Blood 1992; 79: 372-9.
• 33 Cramer EM, Savidge GF, Vainchenker W, Berndt MC, Pidard D, Caen JP, Masse JM, Breton-Gorius J. Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes. Blood 1990; 75: 1220-7.
• 34 Zagursky RJ, Sharp D, Solomon KA, Schwartz A. Quantitation of cellular receptors by a new immunocytochemical flow cytometry technique. Biotechniques 1995; 18: 504-9.
• 35 Wall JE, Buijs Wilt M, Arnold JT, Wang W, White MM, Jennings LK, Jackson CW. A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents. Br J Haematol 1995; 89: 380-5.
• 36 Gawaz M, Neumann FJ, Ott I, Schiessler A, Schömig A. Platelet function in acute myocardial infarction treated with direct angioplasty. Circulation 1996; 93: 229-37.
• 37 Scharf RE, Tomer A, Marzec UM, Teirstein PS, Ruggeri ZM, Harker LA. Activation of platelets in blood perfusing angioplasty-damaged coronary arteries. Arterioscler Thromb 1992; 12: 1475-87.
• 38 Tschöpe D, Schultheiss HP, Kolarov P, Schwippert B, Dannehl K, Nieuwenhuis HK, Kehrel B, Strauer B, Gries FA. Platelet membrane markers are predictive for increased risk of acute ischaemic events after PTCA. Circulation 1993; 88: 37-42.
• 39 Tschöpe D, Rösen P, Esser J, Schwippert B, Nieuwenhuis HK, Kehrel B, Gries FA. Large platelets circulate in an activated state in diabetes mellitus. Semin Thromb Hemost 1991; 17: 433-8.
• 40 Michelson AD, Barnard MR, Hechtmann HB, MacGregor H, Connolly RJ, Loscalzo J, Valeri CR. In vivo tracking of platelets: circulating degranulated platelets rapidly lose surface P-selectin but continue to circulate and function. Proc Natl Acad Sci USA 1996; a 93: 11877-82.
• 41 Collins CE, Cahill MR, Newland AC, Rampton DS. Platelets circulate in an activated state in inflammatory bowel disease. Gastroenterology 1994; 106: 840-5.
• 42 Janes SL, Kyle PM, Redman C, Goodall AH. Flow cytometric detection of activated platelets in pregnant women prior to the development of pre-eclampsia. Thromb Haemost 1995; 74: 1059-63.
• 43 Andrioli G, Ortolani R, Fontana L, Gaino S, Bellavite P, Lechi C, Minuz P, Manzato F, Tridente G, Lechi A. Study of platelet adhesion in patients with uncomplicated hypertension. J Hypertens 1996; 14: 1215-21.
• 44 Laffi G, Cinotti S, Filimberti E, Ciabattoni G, Caporale R, Marra F, Melani L, Grossi A, Carloni V, Gentillini P. Defective aggregation in cirrhosis is independent of in vivo platelet activation. J Hepatol 1996; 24: 436-43.
• 45 Giesberts AN, van Willigen G, Lapetina EG, Akkerman JWN. Regulation of platelet glycoprotein IIb/IIIa (integrin α_IIBβ₃) function via the thrombin receptor. Biochem J 1995; 309: 613-20.
• 46 Michelson AD, Ellis PA, Barnard MR, Matic GB, Viles AF, Kestin AS. Downregulation of the platelet surface glycoprotein Ib-IX complex in whole blood stimulated by thrombin, adenosine diphosphate, or an in vivo wound. Blood 1991; 77: 770-9.
• 47 Michelson AD. Platelet activation by thrombin can be directly measured in whole blood through the use of the peptide GPRP and flow cytometry: methods and clinical studies. Blood Coagul Fibrinolysis 1994; 5: 121-31.
• 48 Frojmovic MM, Milton JG, Gear AL. Platelet aggregation measured in vitro by microscopic and electronic particle counting. Methods Enzymol 1989; 169: 134-49.
• 49 Fabris F, Casonato A, Randi ML, Luzzatto G, De Silvestro G, Ongaro G, Girolami A. The use of fluorescence flow cytometry in the characterisation of Bernard-Soulier syndrome and Glanzmann’s thrombasthenia. Haematologica 1989; 74: 39-44.
• 50 Nurden AT. Human platelet membrane glycoproteins. In: Bloom AL, Forbes CD, Thomas DP, Tuddenham EGD. (eds.) Haemostasis and Thrombosis. Edition III, Churchill Livingstone; Edinburgh: 1994. p 115.
• 51 Gordon N, Thom J, Baker C. and R. Rapid detection of hereditary and acquired platelet storage pool deficiency by flow cytometry. Br J Haematol 1995; 89: 117-23.
• 52 Nagle DL, Karim MA, Woolf EA, Holmgren L, Bork P, Misumi DJ, McGrail SH, Dussault BJ, Perou CM, Boissy RE, Duyk GM, Spritz RA, Moore KJ. Identification and mutation analysis of the complete gene for Chediak-Higashi syndrome. Nat Genet 1996; 14: 307-11.
• 53 Kolluri R, Shehabeldin A, Peacocke M, Lamhonwah AM, TeichertKuliszewska K, Weissmann SM, Siminovitch KA. Identification of WASP mutations in patients with Wiskott-Aldrich syndrome and isolated thrombocytopenia reveals allelic heterogeneity at the WAS locus. Hum Mol Genet 1995; 4: 1119-26.
• 54 Toti F, Satta N, Fressinaud E, Meyer D, Freyssinet JM. Scott syndrome, characterised by impaired transmembrane migration of procoagulant phosphatidylserine and haemorrhagic complications, is an inherited disorder. Blood 1996; 87: 1409-15.
• 55 Stormorken H, Holmsen H, Sund R, Sakariassen KS, Hovig T, Jellum E, Solum NO. Studies on the haemostatic defect in a complicated syndrome. An inverse Scott syndrome platelet membrane abnormality? Thromb Haemost 1995; 74: 1244-51.
• 56 Greinacher A, Admiral J, Dummel V, Vissac A, Kiefel V, Mueller-Eckhardt C. Laboratory diagnosis of heparin-associated thrombocytopenia and comparison of platelet aggregation test, heparin-induced platelet activation test, and platelet factor 4/ heparin enzyme-linked immunosorbent assay. Transfusion 1994; 34: 381-5.
• 57 Lefkovits J, Plow EF, Topol EJ. Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine. N Engl J Med 1995; 332: 1553-9.
• 58 Wittig K, Rothe G, Schmitz G. Inhibition of fibrinogen binding and surface recruitment of GpIIb/IIIa as dose-dependent effects of the RGD-mimetic MK-852. Thromb Haemost 1998; 79: 625-30.
• 59 Ault KA, Rinder HM, Mitchell J, Carmody MB, Vary CP, Hillman RS. The significance of platelets with increased RNA content (reticulated platelets). A measure of the rate of thrombopoiesis. Am J Clin Pathol 1992; 98: 637-46.
• 60 Gawaz M, Fateh-Moghadam S, Pilz G, Gurland HJ, Werdan K. Platelet activation and interaction with leukocytes in patients with sepsis or multiple organ failure. Eur J Clin Invest 1995; 25: 843-51.
• 61 Kienast J, Schmitz G. Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders. Blood 1990; 75: 116-21.
• 62 Ault KA, Knowles C. In vivo biotinylation demonstrates that reticulated platelets are the youngest platelets in circulation. Exp Hematol 1995; 23: 996-1001.
• 63 Romp KG, Peters WP, Hoffman M. Reticulated platelet counts an patients undergoing autologous bone marrow transplantation: an aid in assessing marrow recovery. Am J Hematol 1994; 46: 319-24.
• 64 Rinder HM, Bonan JL, Anandan S, Rinder CS, Rodrigues PA, Smith BR. Noninvasive measurement of platelet kinetics in normal and hypertensive pregnancies. Am J Obstet Gynecol 1994; 170: 117-22.
• 65 Lee LG, Chen CH, Chiu LA. Thiazole orange: a new dye for reticulocyte analysis. Cytometry 1986; 7: 508-17.
• 66 Hirons GT, Fawcett JJ, Crissman HA. TOTO and YOYO: new very bright fluorochromes for DNA content analyses by flow cytometry. Cytometry 1994; 15: 129-40.
• 67 Popov EG, Mejlumian AG, Gavrilov IY, Gabbasov ZA, Pozin E. Evaluation of the ability of intact platelets to accumulate acridine orange. Experientia 1988; 44: 616-8.
• 68 Von dem Borne AE, Decary F. Nomenclature of platelet specific antigens. Hum Immunol 1990; 29: 1-2.
• 69 He R, Reid DM, Jones CE, Shulman NR. Spectrum of Ig classes, specifities, and titers of serum antiglycoproteins in chronic idiopathic thrombocytopenic purpura. Blood 1994; 83: 1024-32.
• 70 Deckmyn H, De Reys S. Functional effects of human antiplatelet antibodies. Semin Thromb Hemost 1995; 21: 46-59.
• 71 Nordhagen R, Flaathen ST. Chloroquine removal of HLA antigens from platelets for the platelet immunofluorescence test. Vox Sang 1985; 48: 156-9.
• 72 Mizumoto Y, Fujimura Y, Nishikawa K, Uchida M, Fukui H, Morii T, Narita N, Kurata Y. Flow cytometric analysis of anti-platelet antibodies in patients with chronic idiopathic thrombocytopenic purpura (ITP) using acid-treated, formalin-fixed platelets. Am J Hematol 1991; 37: 274-6.
• 73 Macchi L, Clofent-Sanchez G, Marit G, Bihour C, Durrieu-Jais C, Besse P, Nurden P, Nurden AT. PAICA: a method for characterizing platelet-associated antibodies – its application to the study of idiopathic thrombocytopenic purpura and to the detection of platelet-bound c7E3. Thromb Haemost 1996; 76: 1020-9.
• 74 Joutsi L, Kekomäki R. Comparison of the direct platelet immunofluores cence test (direct PIFT) with a modified direct monoclonal antibody-specific immobilization of platelet antigens (direct MAIPA) in detection of platelet-associated IgG. Br J Haematol 1997; 96: 204-9.
• 75 Köhler M, Dittmann J, Legler TJ, Lynen R, Humpe A, Riggert J, Neu-meyer H, Pies A, Panzer S, Mayr WR. Flow cytometric detection of platelet-reactive antibodies and application in platelet crossmatching. Transfusion 1996; 36: 250-5.
• 76 Koksch M, Rothe G, Kiefel V, Schmitz G. Fluorescence resonance energy transfer as a new method for the epitope-specific characterisation of antiplatelet antibodies. J Immunol Methods 1995; 187: 53-67.
• 77 Polley MJ, Phillips ML, Wayner E, Nudelman E, Singhal AK, Hakomori SI, Paulson JC. CD62P and endothelial cell-leukocyte adhesion molecule 1 (ELAM-1) recognise the same carbohydrate ligand, sialyl-Lewis x. Proc Natl Acad Science USA 1991; 88: 6224-8.
• 78 Ruf A, Patscheke H. Platelet induced neutrophil activation: platelet expressed fibrinogen induces the oxidative burst in neutrophils by an interaction with CD11c/CD18. Br J Haematol 1995; a 90: 791-6.
• 79 Diacovo TG, de Fougerolles AR, Bainton DF, Springer TA. A functional integrin ligand on the surface of platelets: intercellular adhesion molecule-2. J Clin Invest 1994; 94: 1243-51.
• 80 Amirkhosravi A, Alexander M, May K, Francis DA, Warnes G, Biggerstaff J, Francis JL. The importance of platelets in the expression of monocyte tissue factor antigen measured by a new whole blood flow cytometric assay. Thromb Haemost 1996; 75: 87-95.
• 81 Bath PMW. The routine measurement of platelet size using sodium citrate alone as the anticoagulant. Thromb Haemost 1993; 70: 687-90.
• 82 Bath PMW, Butterworth RJ. Platelet size: measurement, physiology and vascular disease. Blood Coagul Fibrinolysis 1996; 7: 157-61.
• 83 Abrams CS, Ellison N, Budzynski AZ, Shattil SJ. Direct detection of activated platelets and platelet-derived microparticles in humans. Blood 1990; 75: 128-38.
• 84 Warkentin TE, Hayward CP, Boshkov LK, Santos AV, Sheppard JA, Bode AP, Kelton JG. Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia. Blood 1994; 84: 3691-9.
• 85 Yano Y, Kambayashi JI, Kawasaki T, Sakon M. Quantitative determination of circulating platelet microparticles by flow cytometry. Int J Cardiol 1994; 47 (suppl.) S13-19.
• 86 McEver RP, Bennett EM, Martin MN. Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb+IIIa using monoclonal antibodies. J Biol Chem 1983; 258: 5269-75.
• 87 McCarthy DA, Macey MG. Novel anticoagulants for flow cytometric analysis of live leucocytes in whole blood. Cytometr 1996; 23: 196-204.
• 88 Recktenwald D, Prezelin B, Chen CH, Kimura J. Biological pigments as fluorescent labels for cytometry. SPIE 1990; 1206: 106.
• 89 Dean PN, Bagwell CB, Lindmo T, Murphy RF, Salzman GC. Introduction to flow cytometry data file standard. Cytometry 1990; 11: 321-2.
• 90 Kantor AB, Roederer M. FACS analysis of leukocytes. In: Handbook of Experimental Immunology. 5th Ed. 1997
• 91 Schwartz A, Repollet EF, Vogt R, Gratama JW. Standardizing flow cytometry: Construction of a standardized fluorescence calibration plot using matching spectral calibrators. Cytometry 1996; 26: 22-31.
• 92 Michelson AD, Benoit SE, Furman MI, Barnard MR, Nurden P, Nurden AT. The platelet surface expression of glycoprotein V is regulated by two independent mechanisms: proteolysis and a reversible cytoskeletal-mediated redistribution to the surface-connected canalicular system. Blood 1996; 87: 1396-1408.
• 93 Schlossman SF, Boumsell L, Gilks W, Harlan JM, Kishimoto T, Morimoto C, Ritz J, Shaw S, Silverstein R, Springer T, Tedder TF, Todd RF. eds. Leucocyte Typing V: White Cell Differentiation Antigens. Oxford University Press; Oxford: 1995
• 94 von dem Borne AEGK, de Haas M, Simcek S, Porcelijn L, van der Schoot CE. Platelet and neutrophil alloantigens in clinical medicine. Vox Sang 1996; 70 (Suppl. 03) 34-40.