A FLOW CYTOMETRIC METHOD FOR THE DETECTION OF INTRACELLULAR BASIC-PROTEINS IN UNSEPARATED PERIPHERAL-BLOOD AND BONE-MARROW EOSINOPHILS

Eosinophils and their basic proteins play a major role in allergic dis ease and methods are required to monitor their expression in clinical situations. In this article we describe a flow cytometric method for t he detection of intracellular eosinophil cationic protein (ECP) and eo sinophil peroxidase (EPO) in unseparated clinical samples. After fixat ion with parabenzoquinone and permeabilization with n-octyl-beta-D-glu copyranoside, the detection of intracellularly stored proteins was ach ieved using of monoclonal antibodies against ECP (EG1, EG2) and EPO in combination with an FITC-labeled second step antibody. Confocal micro scopy was used to demonstrate the intracellular origin of the fluoresc ent signal, Fixation with parabenzoquinone was superior to a previousl y described protocol using paraformaldehyde, since it reduces non-spec ific binding of FITC to the basic proteins in eosinophils. Fixation an d permeabilization do not alter the light scatter characteristics of e osinophils in contrast to other leukocytes and thus permit gating on e osinophils without prior purification, Furthermore, the procedure does not alter the detection of cell surface antigens on eosinophils and s imultaneous measurements of surface antigens and intracellular protein s is possible. We have used different clinical samples (peripheral blo od, bone marrow cells) to demonstrate differences in the expression of ECP and EPO. We conclude that the detection of inh-acellular eosinoph il proteins by flow cytometry is a rapid, easy and semiquantitative pr ocedure which may be used to study their expression in diseases where eosinophils are involved.