A rapid, automated flow cytometric method to measure activated degranulated platelets by density determination

 

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Summary

Platelet activation is reported to correlate with acute coronary syndromes. A platelet analysis method on the ADVIA^®120 Hematology System provides rapid analysis of platelet density, reported as mean platelet component (MPC) concentration, utilizes routine hematology specimens, requires no pre-treatment, and thirty seconds to generate results. Sub-populations of platelets separated by density gradients showed excellent correlation with the ADVIA 120 MPC parameter (r = 0.997). Platelet activation induced by thrombin treatment resulted in a shift of platelets into the lowest density fraction (d ≤ 1.068 g/mL) with a corresponding reduction in MPC from 24.7 to 20.6 g/dL, N = 4 subjects (p < 0.004). There was also excellent correlation between expression of CD62P measured by fluorescence flow cytometry and the ADVIA 120 Hematology System MPC values (r = 0.85). These results indicate that the ADVIA 120 MPC parameter may be a useful new test for assessing activated platelets and platelet density.

• References

• 1 Mathur A, Robinson MSC, Cotton J, Martin JF. Erusalimsky. Platelet reactivity in acute coronary syndromes: Evidence for differences in platelet behaviour between unstable angina and myocardial infarction. Thromb Haemost 2001; 85: 989-94.
  Article in Thieme ConnectPubMedGoogle Scholar
• 2 Kaplan KL. Laboratory markers of platelet activation. Hemostasis and thrombosis: Basic principles and clinical practice, 3rd Edition. Robert ColmanW., Jack Hirsh, Victor MarderJ., Edwin SalzmanW. J.B. Lippincott Company; Philadelphia: 1994: 1180-96.
  Google Scholar
• 3 Tschoepe D, Schultheiß HP, Kolarov P. et al. Platelet membrane activation markers are predictive for increased risk of acute ischemic events after PTCA. Circulation. 1993; 88: 37-42.
  CrossrefPubMedGoogle Scholar
• 4 Tschoepe D, Roesen P, Esser J. et al. Large platelets circulate in an activated state in diabetes mellitus. Semin Thromb Hemost 1991; 17: 433-8.
  Article in Thieme ConnectPubMedGoogle Scholar
• 5 Lind SE. The bleeding time does not predict surgical bleeding. Blood. 1991; 77: 2547-52.
  PubMedGoogle Scholar
• 6 Ivy AC, Nelson D, Bucher G. The standardization of certain factors in the cutaneous “venostasis” bleeding time technique. J Lab Clin Med 1941; 26: 1812-22.
  PubMedGoogle Scholar
• 7 Hoogendijk EMG, Jenkins CSP, van Wijk EM, Vos J, ten Cate JW. Spontaneous platelet aggregation in cerbrovascular disease II. Further characterisation of the platelet defect. Thromb Haemost 1979; 41: 512-22.
  Article in Thieme ConnectPubMedGoogle Scholar
• 8 Isenhaart CE, Brandt JT. Platelet aggregation studies for the diagnosis of heparin-induced thrombocytopenia. Am J Clin Pathol 1993; 99: 324-30.
  CrossrefPubMedGoogle Scholar
• 9 Ingerman-Wojenski CM, Silver MJ. A quick method of screening platelet dysfunctions using whole blood lumiaggregometry. Thromb Haemost 1984; 51: 154-6.
  Article in Thieme ConnectPubMedGoogle Scholar
• 10 Rao GHR, White JG, Jachomowickz AA, Witkop CJ. An improved method for the extraction of endogenous platelet serotonin. J Lab Clin Med 1976; 87: 129-37.
  PubMedGoogle Scholar
• 11 Sheridan D, Carter C, Kelton JG. A diagnostic test for heparin-induced thrombocytopenia. Blood 1986; 67: 27-30.
  PubMedGoogle Scholar
• 12 Rapold HJ, Grimaudo V, Declerck PJ. et al. Plasma levels of plasminogen activator inhibitor type I, beta-thromboglobulin, and fibrino-peptide A before, during, and after treatment of acute myocardial infarction with alteplase. Blood 1991; 78: 1490-5.
  PubMedGoogle Scholar
• 13 Sadayasu T, Nakashima Y, Yashiro A. et al. Heparin-releasable platelet factor 4 in patients with coronary artery disease. Clin Cardiol 1991; 14: 725-9.
  CrossrefPubMedGoogle Scholar
• 14 van BAOost, van BFEHien-Hagg, Veld-huyzen APM, Timmermans PM, Sixma JJ. Determination of the density distribution of human platelets-Methodological aspects and comparison with other tests for platelet activation. Thromb Haemost 1982; 47: 239-43.
  Article in Thieme ConnectPubMedGoogle Scholar
• 15 Cieslar P, Greenberg JP, Rand ML, Packham MA, Kinlough-Rathbone RL, Mustard JF. Separation of thrombin treated platelets by density gradient centrifugation. Blood 1979; 53: 867-74.
  PubMedGoogle Scholar
• 16 Corash L. Measurement of platelet activation by fluorescence-activated flow cytometry. Blood Cells 1990; 160: 97-108.
  PubMedGoogle Scholar
• 17 van OostB, Hien-Hag IHV, Ans PM, Timmermans PM, Sixma JJ. The effect of thrombin on the density distribution of blood platelets: Detection of activated platelets in the circulation. Blood 1983; 62: 433-40.
  PubMedGoogle Scholar
• 18 van OostBA, Timmermans APM, Sixma JJ. Evidence that platelet density depends on the α-granule content in platelets. Blood 1984; 63: 482-5.
  PubMedGoogle Scholar
• 19 Shattil S, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70: 307-15.
  PubMedGoogle Scholar
• 20 George JN, Pickett EB, Saucerman S. et al. Platelet surface glycoproteins:Studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. J Clin Invest 1986; 78: 340-8.
  CrossrefPubMedGoogle Scholar
• 21 Schmitz G, Rothe G, Ruf A. et al. European Working Group on Clinical Cell Analysis: Consensus protocol for the flow cytometric characterisation of platelet function. Thromb Haemost 1998; 79 (05) 885-96.
  Article in Thieme ConnectPubMedGoogle Scholar
• 22 Michelson AD, Barnard MR, Krueger LA, Frelinger AL, Furman MI. Evaluation of platelet function by flow cytometry. Methods 2000; 21 (03) 259-70.
  CrossrefPubMedGoogle Scholar
• 23 Reimers HJ, Kinlough-Rathbone RL, Caze-nave JP. et al. In vitro and in vivo functions of thrombin-treated platelets. Thromb Haemost 1976; 35: 151-66.
  Article in Thieme ConnectPubMedGoogle Scholar
• 24 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.
  PubMedGoogle Scholar
• 25 Corash L, Tan H, Gralnick H. Heterogeneity of human whole blood platelet subpopulations. I. Relationship between buoyant density, cell volume, and ultrastructure. Blood 1977; 49: 71-87.
  PubMedGoogle Scholar
• 26 DiMinno G, Carbone AM, Mattioll PL, Turco S, Iovine C, Mancini M. Functionally thrombasthenic state in normal platelets following administration of ticlopidine. J Clin Invest 1985; 75: 328-38.
  CrossrefPubMedGoogle Scholar
• 27 Bick RL, Scates SM. Qualitative platelet defects. Lab Med 1992; 23: 95-103.
  CrossrefPubMedGoogle Scholar
• 28 Santoso S, Kunicki TJ, Kroll H, Haberbosch W, Gardemann A. Associated of the platelet glycoprotein Ia C807T gene polymorphism with nonfatal myocardial infarction in younger patients. Blood 1999; 93: 2449-53.
  PubMedGoogle Scholar
• 29 Ethan J, Weiss BA, Bray PF. et al. A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis. N Engl J Med 1996; 334: 1090-4.
  CrossrefPubMedGoogle Scholar
• 30 Harrison P, Ault KA, Chapman S. et al. An interlaboratory study of a candidate reference method for platelet counting. Am J Clin Pathol 2001; 115: 448-59.
  CrossrefPubMedGoogle Scholar
• 31 Corash L, Piomelli S, Chen HC, Seaman C, Gross E. Separation of erythrocytes according to age on a simplied density gradient. J Lab Clin Med 1974; 84: 147-51.
  PubMedGoogle Scholar
• 32 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.
  PubMedGoogle Scholar
• 33 Mie G. Considerations on the optics of turbid media, especially colloidal metal sols. Ann Physik 1908; 25: 377-442.
  PubMedGoogle Scholar
• 34 Kerker M, Cooke D, Chew H, and McNulty P. Light scattering by structured spheres. J Opt Soc Am 1978; 68: 592-601.
  CrossrefPubMedGoogle Scholar
• 35 Kunicka JE, Fischer G, Murphy J, Zelmanovic D. Improved platelet counting using two-dimensional laser light scatter. Am J Clin Pathol 2000; 114: 283-9.
  CrossrefPubMedGoogle Scholar
• 36 Holme S, Murphy S. Platelet storage at 22°C for transfusion: Interrelationship of platelet density and size, medium pH, and viability after in vivo infusion. J Lab Clin Med 1983; 101: 161-74.
  PubMedGoogle Scholar
• 37 Wintrobe’s Clinical Hematology, 9th Ed 1993: 515
  PubMed
• 38 Zelmanovic D, Colella GM, Hetherington E, Chapman ES, Paseltiner L. Automated method and device for identifying and quantifying platelets and for determining platelet activation state using whole blood samples. United States Patent, October 6 1998; Patent Number 5 817: 519
  PubMedGoogle Scholar
• 39 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 applications. Blood Coagul Fibrinolysis 1994; 5: 121-31.
  CrossrefPubMedGoogle Scholar
• 40 Pujol-Moix N, Hernández A, Escolar G, Español I, Martínez-Brotóns F, Mateo J. Platelet ultrastructural morphometry for diagnosis of partial °-storage pool disease in patients with mild platelet dysfunction and/or thrombocytopenia of unknown origin. Study of 24 cases. Haematologica 2000; 85: 620-7.
  PubMedGoogle Scholar
• 41 Brummitt DR, Barker HF. The determination of a reference range for new platelet parameters produced by the Bayer ADVIA 120 full blood count analyser. Clin Lab Haematol 2000; 22: 103-7.
  CrossrefPubMedGoogle Scholar
• 42 Chapman ES, Hetherington EJ, Zelmanovic D. A simple automated method for determining ex vivo platelet activation state. Blood 1996; 88: 2929
  PubMedGoogle Scholar
• 43 Chapman ES, Lerea KM, Kirk R, Sorette MP, Smith SN, Zelmanovic D. Monitoring in vitro and ex vivo platelet activity: Comparison of α-granule release, density distribution, platelet adhesion, and mean platelet component (MPC) concentration. Blood 1998; 92: 3273-68b.
  PubMedGoogle Scholar
• 44 Macey MG, Carty E, Webb L. et al. Use of mean platelet component to measure platelet activation on the ADVIA 120 Haematology System. Cytometry 1999; 38: 250-5.
  CrossrefPubMedGoogle Scholar
• 45 Mohandas N, Kim YR, Tycko DH, Orlik J, Wyatt J, Groner W. Accurate and independent measurement of volume and hemoglobin concentration of individual red cells by laser light scattering. Blood 1986; 68: 506-13.
  PubMedGoogle Scholar
• 46 Mezzano D, Hwang K, Catalano P, Aster RH. Evidence that platelet buoyant density, but not size, correlates with platelet age in man. Am J Hematol 1981; 11: 61-76.
  CrossrefPubMedGoogle Scholar
• 47 Corash L, Costa JL, Shafer B, Donlon JA, Murphy D. Heterogeneity of human blood platelet subpopulation. III. Density-dependent differences in subcellular constituents. Blood. 1984; 64: 185-93.
  PubMedGoogle Scholar