Subtyping lymphocytes in peripheral blood by immunoperoxidase labeling and light scatter/absorption flow cytometry

Lymphocyte subpopulations in a whole-blood sample can be detected by adapting mouse monoclonal antibodies (MAbs) and peroxidase (EC 1.11.1.7) labeling to a flow cytometer equipped with a tungsten-halogen light source and scatter/absorption optics (Technicon H6000). In the optimized cytochemical conditions each cell population generates a distinct, well-separated cluster, for accurate "thresholding" of the surface-antigen negative and positive lymphocyte populations in the presence of other leukocytes. After reaction with MAb, the erythrocytes are lysed, and the lymphocytes and other leukocytes are fixed. Biotinylated anti-mouse IgG, used as a bridge, amplifies the response from the avidin-peroxidase label. Granulocytes and monocytes, which have high endogenous peroxidase activity, and the labeled lymphocytes are stained in a specific amount of hydrogen peroxide plus 4-chloro-1-naphthol in 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid buffer. Accuracy and precision are equivalent to those of flow cytometers that measure immunofluorescence (e.g., Ortho Spectrum III), as demonstrated with OKT3, OKT4, OKT8, OKT11, and Leu 12 MAbs.