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

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.