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Thromb Haemost 1993; 70(04): 659-666
DOI: 10.1055/s-0038-1649645
Original Article
Platelets
Schattauer GmbH Stuttgart

Evaluation of Whole Blood Flow Cytometric Detection of Platelet Bound Fibrinogen on Normal Subjects and Patients with Activated Platelets

Sarah L Janes
1   Department of Haematology, St Georges Hospital Medical School, London SW17, UK
The Haemophilia Centre and Haemostasis Unit, Royal Free Hospital and School of Medicine, London, UK
,
Darren J Wilson
2   Vascular Cell Biology Laboratory, Department of Chemical Pathology & Human Metabolism, Royal Free Hospital School of Medicine, London 2PF, UK
The Haemophilia Centre and Haemostasis Unit, Royal Free Hospital and School of Medicine, London, UK
,
Nicolas Chronos
2   Vascular Cell Biology Laboratory, Department of Chemical Pathology & Human Metabolism, Royal Free Hospital School of Medicine, London 2PF, UK
The Haemophilia Centre and Haemostasis Unit, Royal Free Hospital and School of Medicine, London, UK
,
Alison H Goodall
2   Vascular Cell Biology Laboratory, Department of Chemical Pathology & Human Metabolism, Royal Free Hospital School of Medicine, London 2PF, UK
The Haemophilia Centre and Haemostasis Unit, Royal Free Hospital and School of Medicine, London, UK
› Author Affiliations
Further Information

Publication History

Received 04 December 1992

Accepted after revision 18 May 1993

Publication Date:
05 July 2018 (online)

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Summary

Activated platelets can be detected by measuring platelet-bound fibrinogen in a whole blood, flow cytometric assay, using a fluorescently-conjugated polyclonal antibody.

Fibrinogen binding to unstimulated platelets from normal subjects was low in this assay, as was expression of the CD63 antigen. Single cell counting of samples prepared for flow cytometric analysis showed platelet aggregates do not form during the assay procedure. Immune complexes were not seen, and fibrinogen binding to the platelets was unaffected by the CD32 MAb, IV.3. Artefactual activation of the unfixed samples could be minimised by control of phlebotomy, time and temperature of incubation. Variations in platelet count in the range 140–430 × 109 1-1 and in plasma fibrinogen in the range 2–6 g 1-1 did not affect the assay results.

Comparison of fibrinogen binding with expression of CD63 antigen on normal platelets, stimulated with agonists in vitro, demonstrated that fibrinogen binding detects an earlier stage of platelet activation.

Platelet bound fibrinogen was shown to be sensitive in detecting small numbers of activated platelets in clinical samples in twelve patients on intensive care, four undergoing haemofiltration. The patients had a significantly higher median percentage of circulating platelets with bound fibrinogen (p <0.005), but fibrinogen binding was significantly lower (p <0.02) in response to 10-5 M ADP, compared to twelve age-matched normal Controls.

 

  • References

  • 1 Abrams C, Shattil SJ. Immunological detection of activated platelets in clinical disorders. Thromb Haemostas 1991; 65: 467-473
    PubMedSearch in Google Scholar
  • 2 Shattil SJ, Cunningham M, Hoxie JA. Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry. Blood 1987; 70: 307-315
    CrossrefPubMedSearch in Google Scholar
  • 3 Abrams CS, Ellison N, Budzynski AZ, Shattil SJ. Direct detection of activated platelets and platelet-derived microparticles in humans. Blood 1990; 75: 128-138
    PubMedSearch in Google Scholar
  • 4 Warkentin TE, Powling MJ, Hardisty RM. Measurement of fibrinogen binding to platelets in whole blood by flow cytometry: a micromethod for the detection of platelet activation. Br J Haematol 1990; 76: 387-394
    CrossrefPubMedSearch in Google Scholar
  • 5 Schmitt GW, Moake JL, Rudy CK, Vicks SL, Hamburger RJ. Alterations in hemostatic parameters during hemodialysis with dialyzers of different membrane composition and flow design. Platelet activation and factor VIII-related von Willebrand factor during hemodialysis. Am J Med 1987; 83: 411-418
    CrossrefPubMedSearch in Google Scholar
  • 6 Kelly AB, Hanson SR, Henderson LW, Harker LA. Prevention of heparin-resistant thrombotic occlusion of hollow-fiber hemodialyzers by synthetic antithrombin. J Lab Clin Med 1989; 114: 411-418
    PubMedSearch in Google Scholar
  • 7 Levin RD, Kwaan HC, Ivanovich P. Changes in platelet function during haemodialysis. J Lab Clin Med 1978; 92: 779-786
    PubMedSearch in Google Scholar
  • 8 Goding JW. Monoclonal Antibodies: Principles and Practice. London: Academic Press; 1986. 2.
    Search in Google Scholar
  • 9 Goodall AH. Platelet activation during preparation and storage of concentrates: detection by flow cytometry. Blood Coagul Fibrinol 1991; 2: 377-389
    CrossrefPubMedSearch in Google Scholar
  • 9 Cox AD, Goodall AH. Activation-specific neo-antigens on platelet detected by monoclonal antibodies. In Current Studies in Haematology and Blood Transfusion. Albertini A, Lenfant CL, Mannucci PM, Sixma JJ. (eds) Basel: Karger; 1991: 194-199
  • 11 Bland M. An Introduction to Medical Statistics. Oxford: Oxford Medical Publications; 1990
    Search in Google Scholar
  • 12 George JN, Pickett EB, Saucerman S, McEver RP, Kunicki TJ, Kieffer N, Newman PJ. 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: 340-348
    CrossrefPubMedSearch in Google Scholar
  • 13 Nieuwenhuis HK, van Oosterhout JJG, Rozemuller E, van Iwaarden F, Sixma JJ. Studies with a monoclonal antibody against activated platelets: Evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation. Blood 1987; 70: 838-845
    CrossrefPubMedSearch in Google Scholar
  • 14 Tschoepe D, Rosen P, Esser J, Schwippert B, Nieuwenhuis HK, Kehrel B, Gries FA. Large platelets circulate in an activated state in diabetes mellitus. Semin Thromb Hemostas 1991; 17: 433-438
    Thieme ConnectPubMedSearch in Google Scholar
  • 15 Metzelaar MJ, Sixma JJ, Nieuwenhuis HK. Detection of platelet activation using activation specific monoclonal antibodies. Blood Cells 1990; 16: 85-93
    PubMedSearch in Google Scholar
  • 16 Tschoepe D, Rosen P, Schwippert B, Kehrel B, Schauseil S, Esser J, Gries FA. Platelet analysis using flow cytometric procedures. Platelets 1990; 1: 127-133
    CrossrefPubMedSearch in Google Scholar
  • 17 Lindahl TL, Festin R, Larsson A. Studies of fibrinogen binding to platelets by flow cytometry: An improved method for studies of platelet activation. Thromb Haemostas 1992; 68: 221-225
    PubMedSearch in Google Scholar
  • 18 Sims PJ, Faioni EM, Wiedmer 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 V a and express prothrombinase activity. J Biol Chem 1988; 263: 18205-18212
    PubMedSearch in Google Scholar
  • 19 Michelson AD, Ellis PA, Barnard MR, Matic GB, Viles AF, Kestin AS. Downregulation of the platelet surface glycoprotein Ib-IX com-plex in whole blood stimulated by thrombin, adenosine diphosphate, or in vivo wound. Blood 1991; 77: 770-779
    PubMedSearch in Google Scholar
  • 20 Goodall AH, de Oliveira DomingosM, Chronos N, Janes SL, Wilson DJ. Flow cytometric detection of the redistribution of the GPIb-IX complex on thrombin stimulated platelets is dependent on the type of antibody conjugate used. Blood 1993; 81: 1407-1408
    PubMedSearch in Google Scholar
  • 21 Taylor RR, Sturm M, Vandongen R, Strophair J, Beilin LJ. Whole blood platelet aggregation is not affected by cigarette smoking but is sex-related. Clin Exp Pharmacol Physiol 1987; 14: 665-671
    CrossrefPubMedSearch in Google Scholar