Subtyping lymphocytes in peripheral blood by direct immunoalkaline phosphatase labeling and light scatter/absorption flow cytometric analysis.
Evaluation of blood cells to determine immunologic status is becoming an important clinical application of flow cytometric analysis. For a wider use of immunophenotyping technology in clinical laboratories, the authors developed a rapid method to detect monoclonal antibody-labeled cells using forward light scatter/absorption clinical flow cytometers such as the Technicon H*1 and Technicon H*2 differential complete blood count analyzers. Calf-intestinal alkaline phosphatase was conjugated to mouse monoclonal antibodies (anti-CD2, CD3, CD4, CD8, CD19) for direct immunoenzymatic labeling. The combination of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue-tetrazolium salt in diethanolamine buffer at pH 9.6 was selected as buffer/substrate to yield stable, insoluble, and very intense purplish-blue precipitates on the surface of the cells labeled with monoclonal antibody-alkaline phosphatase conjugates. Endogenous alkaline phosphatase in granulocytes was inhibited with levamisole. Early mild fixation of the white cells permitted incubation at 38 +/- 1 degrees C, which accelerated each step of the reaction without disrupting the cells throughout the procedure. The method is competitive with the direct immunofluorescence whole-blood method used on fluorescence flow cytometers in speed, sensitivity, and accuracy, as demonstrated with alkaline phosphatase-conjugated anti-CD2, CD3, CD4, CD8, CD19 monoclonal antibodies.
Kim
Y R
YR
Research and Development Division, Technicon Instruments Corporation, Tarrytown, New York 10591.
Paseltiner
L
L
Kling
G
G
Yeh
C K
CK
D000469:Alkaline Phosphatase; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique; D006801:Humans; D016130:Immunophenotyping; D008027:Light; D016131:Lymphocyte Subsets / Q000276:immunology*; D012044:Regression Analysis; D012542:Scattering, Radiation; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000469:Alkaline Phosphatase