SIMULTANEOUS DETECTION OF IGM AND IGG ANTIBODIES AGAINST PLATELETS, LYMPHOCYTES, AND GRANULOCYTES BY FLOW-CYTOMETRY

Background: Typically, immunofluorescence-based assays to detect antib odies against blood cells consist of separate tests on cell suspension s enriched for the individual populations. Here, we present a three-co lor now cytometric assay that allows the simultaneous detection of IgM and IgG antibodies against platelets, lymphocytes and granulocytes, u sing a mixed suspension of these three cell populations. Materials and Methods: Platelets, lymphocytes, and granulocytes are isolated and re constituted in equal proportions, followed by incubation steps with se rum and antihuman IgM and IgG conjugates. A mixture of laser dye solut ion 751 and propidiumiodide is added to the cells 10 min prior to flow cytometry in order to exclude residual erythrocytes and damaged and/o r dead nucleated cells during data analysis on the basis of their nonr eactivity and very strong reactivity with these red-fluorescent dyes, respectively. Predefined, fixed light scatter and fluorescence (FL) re gion and marker settings or definitions for cluster algorithms are use d in all experiments to identify the individual cell populations and t o discriminate positive from negative immunofluorescence. This approac h obviates the need for subjective adjustments of marker settings. Res ults: We demonstrated the application of this assay using conventional list mode data analysis software (CellQuest(TM)) and cluster analysis software (Attractors(TM)). This flow cytometric assay is more sensiti ve than conventional assays (e.g. complement-dependent cytotoxicity or microscopic immunofluorescence assays) for the detection of alloantib odies against platelets, lymphocytes, and granulocytes. Conclusion: Th e simultaneous flow cytometric detection of IgM and IgG antibodies aga inst platelets, lymphocytes, and granulocytes is a rapid, sensitive an d objective assay which is useful for detection of alloantibodies and crossmatching in transfusion medicine.