Immunoglobulin and enzyme-conjugated dextran polymers enhance u-PAR staining intensity of carcinoma cells in peripheral blood smears

The presence of disseminated carcinoma cells in bone marrow and peripheral blood has prognostic importance in patients with carcinomas. Much evidence indicates that dissemination of tumor cells may depend on activation of a v ariety of degradative enzymes. A strong positive correlation has been shown between the expression of tumor cell proteases and tumor invasion. Therefo re, phenotypic characterization of disseminated carcinoma cells for express ion of protease activators might define the invasive potential of the cells . We present an immunocytochemically enhanced staining method that allows p henotyping of disseminated carcinoma cells in bone marrow and peripheral bl ood smears. In the first step, the cells were incubated with antibodies aga inst urokinase plasminogen activator receptor (u-PAR) and subsequently with secondary antibodies conjugated to peroxidase-labeled dextran polymers. A brown color reaction was developed with diaminobenzidine as chromogen. In t he second step, the cells were incubated with alkaline phosphatase-conjugat ed murine monoclonal antibodies against a common cytokeratin epitope and a red color reaction was developed with new fuchsin as substrate. This method allows simultaneous and unambiguous immunolabeling of intracellular cytoke ratin and of u-PAR intracellularly and on the surface of carcinoma cells. T his novel approach can be used for detection and phenotyping of carcinoma c ells in blood smears for u-PAR or, presumably, for any other heterogeneousl y expressed antigen on the surface of the detected cells.