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 vari ety of diseases, We have developed a flow cytometry-based assay for monitor ing the presence of such conjugates, By preloading the sampling tubes with 1/8 volume of 4% paraformaldehyde, basal levels of platelet-leukocyte conju gates was stable for at least 48h 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 wh ole blood from healthy individuals, platelets were found adherent to 3.2% o f the lymphocytes, 4.1% of the monocytes and to 2.5% of the granulocytes, T he 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 (AD P, TRAP) stimulation, the platelet adhesion to monocyte and granulocytes in creased several-fold, Microscopy verified the flow cytometry results and re vealed that platelet adhered to the leukocytes as platelet-platelet aggrega tes 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 ass ay well suited for clinical studies, the recommended approach may be a valu able tool in establishing the clinical significance of circulating platelet -leukocyte conjugates.