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Related Experiment Videos

A sensitive flow cytometric assay for circulating platelet-leucocyte aggregates

N Li1, A H Goodall, P Hjemdahl

  • 1Department of Laboratory Medicine, Karolinska Hospital, Stockholm, Sweden.

British Journal of Haematology
|January 31, 1998
PubMed
Summary

A new whole blood flow cytometry method accurately measures in vivo platelet-leucocyte aggregates (PLAs). This technique avoids artifacts, revealing preformed aggregates and their agonist-driven formation mechanisms.

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Area of Science:

  • Immunology
  • Hematology
  • Cell Biology

Background:

  • Platelet-leucocyte aggregates (PLAs) are implicated in various inflammatory and thrombotic conditions.
  • Accurate quantification of in vivo PLAs is crucial for understanding their role in disease.
  • Existing methods often introduce artifacts due to fixation or centrifugation.

Purpose of the Study:

  • To describe a novel whole blood flow cytometric method for quantifying in vivo PLAs without fixation or centrifugation.
  • To validate the method by assessing artifactual increases and agonist-induced PLA formation.
  • To investigate the mechanisms underlying PLA formation using blocking antibodies.

Main Methods:

  • Developed a whole blood flow cytometry assay for platelet-leucocyte aggregate (PLA) analysis.

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  • Compared results with and without fixation, centrifugation, and red blood cell lysis.
  • Stimulated unfixed whole blood with agonists like ADP, thrombin, platelet activating factor (PAF), and N-formyl-methionyl-leucyl-phenylalanine (fMLP).
  • Utilized blocking monoclonal antibodies (MAbs) to probe ligand-receptor interactions.

    • Main Results:

    • The developed method showed 15.3 +/- 8.5% PLAs in healthy volunteers, suggesting preformed aggregates in vivo.
    • Fixation and centrifugation significantly increased artefactual PLA counts (3-5 fold).
    • Agonists (ADP, thrombin, PAF, fMLP) dose-dependently increased PLA formation in unfixed blood.
    • Blocking antibodies revealed distinct mechanisms for PLA formation mediated by different agonists, involving P-selectin and GPIIb/IIIa interactions.

    Conclusions:

    • The described whole blood flow cytometry method provides a reliable assessment of in vivo platelet-leucocyte aggregates.
    • The method minimizes artifacts, allowing for accurate studies of PLA formation and function.
    • Findings elucidate the specific molecular mechanisms and receptor systems involved in agonist-induced PLA formation.