Thromb Haemost 2003; 89(06): 1004-1015
DOI: 10.1055/s-0037-1613402
Platelets and Blood Cells
Schattauer GmbH

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

Sabrinah E. Chapman
1   Bayer Diagnostics, New York, U.S.A
,
Martin Sorette
1   Bayer Diagnostics, New York, U.S.A
,
Edward Hetherington
1   Bayer Diagnostics, New York, U.S.A
,
David Zelmanovic
1   Bayer Diagnostics, New York, U.S.A
,
Gail Kling
1   Bayer Diagnostics, New York, U.S.A
,
Jacques Dugailliez
1   Bayer Diagnostics, New York, U.S.A
,
Nuria Pujol-Moix
1   Bayer Diagnostics, New York, U.S.A
,
David Okrongly
1   Bayer Diagnostics, New York, U.S.A
› Author Affiliations
Further Information

Publication History

Received 13 January 2003

Accepted after revision 14 March 2003

Publication Date:
08 December 2017 (online)

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.
  • 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.
  • 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.
  • 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.
  • 5 Lind SE. The bleeding time does not predict surgical bleeding. Blood. 1991; 77: 2547-52.
  • 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.
  • 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.
  • 8 Isenhaart CE, Brandt JT. Platelet aggregation studies for the diagnosis of heparin-induced thrombocytopenia. Am J Clin Pathol 1993; 99: 324-30.
  • 9 Ingerman-Wojenski CM, Silver MJ. A quick method of screening platelet dysfunctions using whole blood lumiaggregometry. Thromb Haemost 1984; 51: 154-6.
  • 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.
  • 11 Sheridan D, Carter C, Kelton JG. A diagnostic test for heparin-induced thrombocytopenia. Blood 1986; 67: 27-30.
  • 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.
  • 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.
  • 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.
  • 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.
  • 16 Corash L. Measurement of platelet activation by fluorescence-activated flow cytometry. Blood Cells 1990; 160: 97-108.
  • 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.
  • 18 van OostBA, Timmermans APM, Sixma JJ. Evidence that platelet density depends on the α-granule content in platelets. Blood 1984; 63: 482-5.
  • 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.
  • 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.
  • 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.
  • 22 Michelson AD, Barnard MR, Krueger LA, Frelinger AL, Furman MI. Evaluation of platelet function by flow cytometry. Methods 2000; 21 (03) 259-70.
  • 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.
  • 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.
  • 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.
  • 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.
  • 27 Bick RL, Scates SM. Qualitative platelet defects. Lab Med 1992; 23: 95-103.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 33 Mie G. Considerations on the optics of turbid media, especially colloidal metal sols. Ann Physik 1908; 25: 377-442.
  • 34 Kerker M, Cooke D, Chew H, and McNulty P. Light scattering by structured spheres. J Opt Soc Am 1978; 68: 592-601.
  • 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.
  • 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.
  • 37 Wintrobe’s Clinical Hematology, 9th Ed 1993: 515
  • 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
  • 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.
  • 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.
  • 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.
  • 42 Chapman ES, Hetherington EJ, Zelmanovic D. A simple automated method for determining ex vivo platelet activation state. Blood 1996; 88: 2929
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.