The analysis of cell function can provide relevant clinical information that cannot be acquired from static cellular parameters, such as the expression of surface antigens. Since these considerations are particularly relevant in transplantation medicine and diseases of the immune system, many investigations have focused on functional analysis of the lymphocyte. Virtually every event that occurs during the process of lymphocyte activation can be measured by flow cytometry, but determinations of tyrosine phosphorylation, calcium flux, oxidative metabolism, neoantigen expression, and cellular proliferation have the greatest clinical potential at this time.Tyrosine phosphorylation can be measured within the cell by multiparametric flow cytometry and labeled antiphosphotyrosine monoclonal antibodies, or within activated cell lysates with colored microbeads labeled with monoclonal antibodies specific for different tyrosine kinase substrates Intracellular calcium flux is measured with ratiometric cacium ion indicators whose spectral characteristics change with calcium ion binding. Calcium flux has been used to study platelet activation in response to different agonists and lymphocyte activation in viral infection and other diseases. Flow cytometric measurement of the oxidative burst in neutrophils has been used as a screening test for chronic granulomatous disease. The most common technique for this purpose uses a nonfluorescing dye (dihydrorhodamine-123) that is selectively concentrated in the mitochondria and is oxidized to a brightly fluorescent compound (rhodamine-123) during the normal oxidative burst.Lymphocyte neoantigens are surface or intracellular proteins, including cytokines, that are up-regulated during lymphocyte activation. The detection of these substances may become one of the most important flow cytometric assays.(15.)
