Evaluation of circulating platelet-leukocyte conjugates: a sensitive flow cytometric assay well suited for clinical studies.
Circulating platelet-leukocyte conjugates are found in patients with a variety of diseases. We have developed a flow cytometry-based assay for monitoring the presence of such conjugates. By preloading the sampling tubes with 1/8 volume of 4% paraformaldehyde, basal levels of platelet-leukocyte conjugates was stable for at least 48 h after blood sampling. The leukocytes were discriminated from the other cells in fixed whole blood by binding of anti-CD45, and no lysing, washing, or centrifugation steps were required. In whole blood from healthy individuals, platelets were found adherent to 3.2% of the lymphocytes, 4.1% of the monocytes and to 2.5% of the granulocytes. The corresponding values for mean number of platelets per leukocyte were 1.3 x 10(-2), 2.5 x 10(-2) and 1.6 x 10(-2), respectively. Upon in vitro agonist (ADP, TRAP) stimulation, the platelet adhesion to monocyte and granulocytes increased several-fold. Microscopy verified the flow cytometry results and revealed that platelet adhered to the leukocytes as platelet-platelet aggregates and not as single platelets as the agonist concentration increased. At high concentrations of TRAP, the strong agonist, large multiconjugates with several granulocytes per platelet-platelet aggregate evolved. Being an assay well suited for clinical studies, the recommended approach may be a valuable tool in establishing the clinical significance of circulating platelet-leukocyte conjugates.
Hagberg
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Ullevaal University Hospital, Oslo, Norway. ingeranne.hagberg@ulleval.no
Lyberg
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D000244:Adenosine Diphosphate / Q000494:pharmacology; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005404:Fixatives / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods; D005557:Formaldehyde / Q000494:pharmacology; D006098:Granulocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D006801:Humans; D017493:Leukocyte Common Antigens / Q000097:blood; D007962:Leukocytes / Q000166:cytology* / Q000276:immunology / Q000378:metabolism*; D018950:Lipopolysaccharide Receptors / Q000097:blood; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D019007:P-Selectin / Q000276:immunology / Q000378:metabolism; D010973:Platelet Adhesiveness / Q000187:drug effects; D010979:Platelet Function Tests / Q000295:instrumentation / Q000379:methods / Q000592:standards; D011108:Polymers / Q000494:pharmacology; D011506:Proteins / Q000494:pharmacology; D018179:Receptors, Thrombin
D000911:Antibodies, Monoclonal; D005404:Fixatives; D018950:Lipopolysaccharide Receptors; D019007:P-Selectin; D011108:Polymers; D011506:Proteins; D018179:Receptors, Thrombin; D005557:Formaldehyde; D000244:Adenosine Diphosphate; D017493:Leukocyte Common Antigens; C003043:paraform