Patent ID: 6667149
Filing Date: 2003-12-23
Classification: G01N

Abstract:
A procedure for the analysis of functional activation of leukocytes and platelets produced in vivo or induced in vitro, based on i) specific stabilization of cytoplasmic membrane proteins, said proteins being expressed on the surface of said leukocytes and platelets as a consequence of said functional activation, and are rapidly cleaved from the cytoplasmic membrane, by specific cytoplasmic membrane proteases expressed by the same cell through the inhibition of said membrane proteases, ii) identification of subpopulations of said leukocytes and platelets and iii) detection of said specifically stabilized cytoplasmic membrane proteins through quantitative cytometric techniques without manipulation of cells, comprising the steps of:(a) sequentially incubating a sample containing subpopulations of leukocytes or platelets by using an inhibitor specific for a membrane protease and a plurality of different monoclonal antibodies each conjugated with a different fluorochrome, which include: i) at least one fluorochrome-labeled monoclonal antibody specific for a stabilized cytoplasmic membrane protein; and ii) at least a second monoclonal antibody conjugated to a different fluorochrome, specific for the identification of a non-stabilized cytoplasmic membrane protein, which binds and identifies a non-stabilized cytoplasmic membrane protein whose pattern of expression is specific for a subpopulation leukocytes or platelets from the sample; (b) measuring by quantitative cytometry the fluorescence emissions of the fluorochromes conjugated with: i) each of the monoclonal antibodies bound to a stabilized membrane protein and ii) each of the monoclonal antibodies bound to a non-stabilized cytoplasmic membrane protein whose pattern of expression is specific for a subpopulation of leukocytes and platelets from the sample, as defined in step (a); and (c) analyzing the measurements obtained in step (b), by i) identifying each subpopulation of leukocytes and platelets present in the sample based on the fluorescence emissions of the fluorochromes conjugated with the monoclonal antibodies specific for non-stabilized-cytoplasmic membrane proteins whose pattern of expression is also specific for a given subpopulation of leukocytes and platelets and ii) determining the levels of expression of each stabilized cytoplasmic membrane protein measured as defined in step (b) for each of the subpopulations of leukocytes and platelets present in the sample and identified as defined above.