title: The new violet laser dye, Krome Orange, allows an optimal polychromatic immunophenotyping based on CD45-KO gating.
abstract: BACKGROUND
  Polychromatic immunophenotyping improves characterization of leukocyte subpopulations and their malignant counterparts. However, the lack of various fluorochrome-labeled monoclonal antibodies (MoAbs) hinders the formation of multi-color panels. CD45 appears to be an important MoAb for immunophenotyping of these cells. Plotted against the side scatter, CD45 provides immunological cell differentiation and the ability to recognize various normal and malignant leukocyte subpopulations. CD45 is commonly used and labeled with various fluorochromes and as a result, is incorporated in multi-color panels as a conjugate of less available fluorochromes, such as the violet laser dyes. However, these dyes (e.g. Pacific Orange/PO) often possess low fluorescence intensity, which may be too weak to differentiate between populations. The new organic dye Krome Orange (KO, emission at 528 nm) appears to be a more intense violet laser dye, serving as an alternative to PO.
  
  
  METHODS
  Intensities of CD45 conjugated with FITC, PE, ECD, PE-Cy5, PE-Cy7, PO and KO were tested in different cell sources. Various lineage markers were sequentially back gated on CD45-KO to identify subpopulations. A 10-color MoAb panel for determination of aberrancies in small cell samples was composed to test specificity of CD45-KO.
  
  
  CONCLUSIONS
  We showed in various fixed and unfixed cells from different sources that KO is a suitable fluorochrome with a significantly higher quantum yield than PO and is even brighter than other violet laser dyes (e.g. Pacific Blue). CD45-KO/SS enables us to distinguish and characterize various normal and malignant leukocyte subpopulations. By using a 10-color MoAb panel to screen on aberrancies, we showed that CD45-KO provides reliable immunophenotyping within small amounts of cells and thereby improves the quality of 10-color stainings.
authors.affiliation: Department of Laboratory Medicine, Laboratory of Hematology, Radboud University Nijmegen Medical Center, Geert Grooteplein 8, 6525 GA Nijmegen, The Netherlands. f.preyers@labgk.umcn.nl
authors.forename: Frank W M B
authors.initials: FW
authors.lastname: Preijers
authors.forename: Erik
authors.initials: E
authors.lastname: Huys
authors.forename: Marij
authors.initials: M
authors.lastname: Leenders
authors.forename: Laura
authors.initials: L
authors.lastname: Nieto
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Gautherot
authors.forename: Bijan
authors.initials: B
authors.lastname: Moshaver
mesh_terms: D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D018709:Statistics, Nonparametric
chemical_list: D005456:Fluorescent Dyes; D017493:Leukocyte Common Antigens