An immunogold silver staining method for the light microscopic analysis of blood lymphocyte subsets with monoclonal antibodies

This paper introduces an immunogold silver staining procedure for the identification of human lymphocyte subpopulations in optical microscopy. The described procedure is performed on cells in suspension (peripheral mononuclear cells or blood buffy-coat cells) and uses conventional antilymphocytic monoclonal antibodies (OKT3, OKT4, OKT8, OKIa1) followed by gold-labeled secondary antibodies (GAM G30 or G40) and silver sensitization. It ends with the preparation of permanent records (smears or cytocentrifuge preparations) which are counterstained with standard panoptic Wright or May-Grünwald Giemsa stain. In all cases, the surface antigens appear as numerous black dots on the lymphocytes, with a strong labeling reaction that allows one to clearly distinguish between negative and positive cells. The comparison with immunofluorescence microscopy in normal individuals and in patients indicates that this new immunostaining technic is specific and more sensitive. It has the advantages of using small amounts of blood (1 mL per test), of being rapid (three hours), and of allowing the simultaneous immunophenotypic and morphologic evaluation on smears. This makes it suitable for the analysis of other biologic specimens not already accessible with immunofluorescence. The labeled preparations apparently can be stored indefinitely, and are thus useful for longitudinal studies in the same patient. Finally, the method does not require sophisticated equipment and may be subjected to automated analysis.