A method of analyzing procoagulant activity in monocytes at single cell level

Procoagulant activity (PCA) of monocytes is known to play a pivotal role in a variety of physiologic and pathophysiologic processes, such as disseminated intravascular coagulation, atherosclerosis, arterial and venous thromboembolism, cancer-related hypercoagulability and immunopathologies. Until now, PCA has been studied by clotting assays of a whole cell population or at single cell level by analyzing tissue factor antigen, the protein that initiates PCA but does not always correlate with it. Here, we describe a new simple flow cytometric method that allows the PCA of monocytes to be studied at a single cell level by quantifying the fibrin formed around the cells in suspension.

Purified fibrinogen was tagged with FITC and added to a recalcified developer plasma containing suitable amounts of heparin in order to inhibit the expansion of clotting, thus limiting the formation of fibrin to the surface of cells with PCA. With appropiate amounts of heparin, in 10 min, large sea urchin-like cells with fibrin needles around some monocytes were formed and, after fixation, cytofluorimetrically analyzed.

Blood mononuclear cells isolated and immediately analyzed showed less than 0.1% sea urchin cells. Adherence alone, lipopolysaccharides or ionomycin stimulated expression of PCA in a dose- and time-dependent relationship: after 30 min, 1-3% of the MNC showed PCA, and after 20 h this reached 5-10%. Density separation of monocytes showed that different stimulators act on different maturation stages. Subjects with diabetes express more monocytes with PCA than normal subjects after 30 min stimulation.

This method allows PCA analysis of monocytes at single cell level and requires only a low number of cells. The signal produced by the fluorescent fibrin is strong and easily analyzed by flow cytometry. The method is suitable for analyzing blood from patients with different pathologies and many conditions under different stimuli.