title: Fluorescence-intensity multiplexing: simultaneous seven-marker, two-color immunophenotyping using flow cytometry.
abstract: BACKGROUND
  Conventional immuno-based multiparameter flow cytometric analysis has been limited by the requirement of a dedicated detection channel for each antibody-fluorophore set. To address the need to resolve multiple biological targets simultaneously, flow cytometers with as many as 10-15 detection channels have been developed. In this study, a new Zenon immunolabeling technology is developed that allows for multiple antigen detection per detection channel using a single fluorophore, through a unique method of fluorescence-intensity multiplexing. By varying the Zenon labeling reagent-to-antibody molar ratio, the fluorescence intensity of the antibody-labeled cellular targets can be used as a unique identifier. Although demonstrated in the present study with lymphocyte immunophenotyping, this approach is broadly applicable for any immuno-based multiplexed flow cytomety assay.
  
  
  METHODS
  Lymphocyte immunophenotyping of 38 clinical blood specimens using CD3, CD4, CD8, CD16, CD56, CD19, and CD20 antibodies was performed using conventional flow cytometric analysis and fluorescence-intensity multiplexing analysis. Conventional analysis measures a single antibody-fluorophore per photomultiplier tube (PMT). Fluorescence-intensity multiplex analysis simultaneously measures seven markers with two PMTs, using Zenon labeling reagent-antibody complexes in a single tube: CD19, CD4, CD8, and CD16 antibodies labeled with Zenon Alexa Fluor 488 Mouse IgG(1) labeling reagent and CD56, CD3, and CD20 antibodies labeled with Zenon R-Phycoerythrin (R-PE) Mouse IgG(1) or IgG(2b) labeling reagents.
  
  
  RESULTS
  The lymphocyte immunophenotyping results from fluorescence-intensity multiplexing using Zenon labeling reagents in a single tube were comparable to results from conventional flow cytometric analysis.
  
  
  CONCLUSIONS
  Simultaneous evaluation of multiple antigens using a single fluorophore can be performed using antibodies labeled with varying ratios of a Zenon labeling reagent. Labeling two sets of antibodies with different Zenon labeling reagents can generate characteristic and distinguishable multivariate patterns. Combining multiple antibodies and fluorescent labels with fluorescence intensity multiplexing enables the resolution of more cellular targets than detection-channels, allowing sophisticated multiparameter flow cytometric studies to be performed on less complex 2- or 3-detection-channel flow cytometers. For typical biological samples, approximately 2-4 cellular targets per detection channel can be resolved using this technique.
authors.affiliation: Flow Cytometry Department, Molecular Probes, 29851 Willow Creek Road, Eugene, OR 97402, USA. jolene.bradford@probes.com
authors.forename: Jolene A
authors.initials: JA
authors.lastname: Bradford
authors.forename: Gayle
authors.initials: G
authors.lastname: Buller
authors.forename: Michael
authors.initials: M
authors.lastname: Suter
authors.forename: Michael
authors.initials: M
authors.lastname: Ignatius
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Beechem
mesh_terms: D018941:Antigens, CD19 / Q000096:biosynthesis; D018951:Antigens, CD20 / Q000096:biosynthesis; D017252:CD3 Complex / Q000096:biosynthesis; D015704:CD4 Antigens / Q000096:biosynthesis; D019002:CD56 Antigen / Q000096:biosynthesis; D016827:CD8 Antigens / Q000096:biosynthesis; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D016130:Immunophenotyping / Q000295:instrumentation / Q000379:methods*; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology; D017452:Receptors, IgG / Q000096:biosynthesis; D013997:Time Factors
chemical_list: D018941:Antigens, CD19; D018951:Antigens, CD20; D017252:CD3 Complex; D015704:CD4 Antigens; D019002:CD56 Antigen; D016827:CD8 Antigens; D017452:Receptors, IgG