Immunogold staining: an alternative method for lymphocyte subset enumeration. Comparison with immunofluorescence microscopy and flow cytometry.

An immunogold staining procedure for light microscopic enumeration of peripheral blood lymphocyte subsets defined by monoclonal antibodies (OKT3, OKT4, OKT8, OKIa1, Leu 1, Leu 4, Leu 2a, Leu 3a, Leu 10, Leu 12, B1) is described. It uses colloidal gold-labeled goat anti-mouse Ig (GAM G40 and GAM G30) as second layer and a methyl-green pyronin counterstain. Performed on small volumes of blood without sophisticated laboratory equipment, this method allows accurate cell type recognition and permanent records, essential for longitudinal observations. By enumerating the gold particles on positively labeled cells, it was shown that the staining reactivity depended on the monoclonal antibody used. Lymphocytes reacting with OKT8 or OKIa1 or B1 exhibited the strongest labeling whereas OKT4+ cells were weakly labeled. When compared with flow cytometry analysis in healthy subjects, the accuracy, precision and sensitivity of both methods were very similar. Similarly, a close correlation (97%) was found between immunogold staining and immunofluorescence microscopy in 35 patients with various diseases suggesting that immunogold staining may be useful in a clinical context.