Evaluation of circulating platelet–leukocyte conjugates: a sensitive flow cytometric assay well suited for clinical studies

Abstract

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 1.3·10–2, 2.5·10 -2 and 1.6·10– -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.