title: Rapid single-step method for flow cytometric detection of surface and intracellular antigens using whole blood.
abstract: Fixation/permeabilization methods used for the detection of intracellular antigens by flow cytometry often result in the destruction of cellular morphology and surface immunoreactivity, properties useful in flow cytometry for the characterization of cells in heterogeneous populations. In addition, a majority of these methods are incompatible with whole blood and require that peripheral blood leukocytes (PBLs) be purified prior to fixation. This article describes a new technique for the rapid detection of both intracellular and cell surface antigens, while preserving cell morphology, through the use of a single-step fixation/permeabilization reagent, ORTHO PermeaFix (OPF). OPF is compatible with whole blood, allowing for the direct preparation of PBLs without prior cell separation. An additional red blood cell lysing reagent was not required because RBC lysis occurred upon resuspension of OPF-treated whole blood samples in isotonic solution. Discrimination of leukocyte populations by light scatter after OPF treatment was comparable to matched unfixed live cells. In addition, absolute lymphocyte and white blood cell (WBC) counts were not significantly affected when OPF-treated cells were compared with unfixed cells. Treatment of whole blood from 7 normal donors showed no significant difference in percentage of cells positive for CD2, CD3, CD4, CD8, CD16, or CD19 between fixed and unfixed samples when cells were stained before fixation, and no difference in CD3, CD4, CD8, CD16, or CD19 percentages when cells were stained following fixation. Monoclonal antibodies specific for intracellular antigens located at various sites within the cell were tested on fixed samples. OPF-treated peripheral blood lymphocytes showed greater than 95% reactivity for the inner mitochondrial membrane protein bcl-2, and the cytoskeletal cytoplasmic protein vimentin. TIA-1, a cytolytic granule-associated protein, showed differential reactivity within lymphocyte subsets, from a low of 8 +/- 2% in CD4+ cells to 89 +/- 6% in CD16+ cells, when whole blood from five normal donors was fixed and stained. Reh cells treated with OPF showed greater than 95% reactivity for the internuclear protein TdT. A comparison of OPF with two other fixation/permeabilization procedures, 1% paraformaldehyde followed by 45% ethanol and 0.25% paraformaldehyde followed by 0.2% Tween 20, showed that only OPF could be used both prior to or following cell surface staining with no effect on antigen detection while allowing optimal detection of all of the intracellular antigens tested.
authors.affiliation: Immunocytometry Department, Ortho Diagnostic Systems, Inc., Raritan, NJ 08869-0606, USA.
authors.forename: C
authors.initials: C
authors.lastname: Francis
authors.forename: M C
authors.initials: MC
authors.lastname: Connelly
mesh_terms: D000941:Antigens / Q000097:blood*; D015703:Antigens, CD / Q000097:blood; D000954:Antigens, Surface / Q000097:blood; D015415:Biomarkers / Q000097:blood; D001798:Blood Proteins / Q000032:analysis; D002463:Cell Membrane Permeability / Q000187:drug effects*; D005404:Fixatives / Q000494:pharmacology*; D005434:Flow Cytometry / Q000379:methods*; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007424:Intracellular Fluid / Q000276:immunology*; D007958:Leukocyte Count / Q000379:methods*; D016131:Lymphocyte Subsets / Q000187:drug effects / Q000276:immunology*; D009930:Organic Chemicals; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D000941:Antigens; D015703:Antigens, CD; D000954:Antigens, Surface; D015415:Biomarkers; D001798:Blood Proteins; D005404:Fixatives; C103844:ORTHO permeafix; D009930:Organic Chemicals