title: Detection of membrane and intracellular antigens by flow cytometry following ORTHO PermeaFix fixation.
abstract: In the present study, we explored the suitability of a new cell fixative (ORTHO PermeaFix, OPF) for the detection by flow cytometry of intracellular molecules while preserving the cell surface immunoreactivity, scatter features and morphology. The effect of OPF was investigated on whole blood of ten normal donors, and on separated blasts of 17 leukemic patients. OPF fixation for 45 min to 24 h maintained the morphology of lymphoid cells with minimal cellular distortion and scatter changes, and only slightly modified cell surface immunoreactivity. For at least 1 week following fixation, the cells were still suitable for immunostaining with monoclonal antibodies that recognize the main lymphoid populations. These included CD3, CD4 and CD8 for T-cell subsets, CD19 and CD16 for B lymphocytes and NK cells, and CD45 for leukocyte common antigen (LCA). The OPF fixation of leukemic cells allowed the simultaneous detection of nuclear TdT in conjunction with membrane CD19, and with membrane and/or cytoplasmic CD22 in common-ALL, as well as with cytoplasmic CD3 in T-ALL cases. Our findings suggest that with the introduction of this new fixative into the routine laboratory service, a number of convenient and practical arrangements can be made which increase the efficiency of immunodiagnosis. Small laboratories with no inhouse flow-cytometric facilities can now accumulate OPF-treated whole blood samples for at least 3-4 days and send these to reference laboratories. In addition, the immunodiagnosis of acute leukemia is greatly facilitated by combination staining for membrane and intracellular antigens both at diagnosis and when the analysis of minority populations is warranted for detecting minimal disease.
authors.affiliation: Department of Haematology, Verona University School of Medicine, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Pizzolo
authors.forename: C
authors.initials: C
authors.lastname: Vincenzi
authors.forename: G
authors.initials: G
authors.lastname: Nadali
authors.forename: D
authors.initials: D
authors.lastname: Veneri
authors.forename: F
authors.initials: F
authors.lastname: Vinante
authors.forename: M
authors.initials: M
authors.lastname: Chilosi
authors.forename: G
authors.initials: G
authors.lastname: Basso
authors.forename: M C
authors.initials: MC
authors.lastname: Connelly
authors.forename: G
authors.initials: G
authors.lastname: Janossy
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D000951:Antigens, Neoplasm / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis*; D002467:Cell Nucleus / Q000276:immunology; D003593:Cytoplasm / Q000276:immunology; D005404:Fixatives / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000276:immunology*
chemical_list: D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D005404:Fixatives