title: An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia.
abstract: BACKGROUND
  ZAP-70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B-cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP-70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations.
  
  
  METHODS
  A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4-17) to measure intracellular phospho-epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP-70 expression in positive T- or NK-cells as compared to negative B-cells in peripheral blood samples. A number of commercially available anti-ZAP-70 antibody-conjugates were screened using this methodology, and the antibody-conjugate showing the best performance was chosen to develop a four-color, five antibody assays to measure ZAP-70 levels in whole blood specimens.
  
  
  RESULTS
  Using the optimized fixation and permeabilization method, improvement in assay performance (signal-to-noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set-up protocols, we obtained both intra- and interlaboratory reproducibility in the analysis of ZAP-70 expression in whole blood samples from normal and CLL patients.
  
  
  CONCLUSIONS
  The development of a sensitive, specific and highly reproducible ZAP-70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology-based cutoff points for clinical outcomes must next be established before ZAP-70 protein analysis can be routinely implemented in the clinical laboratory.
authors.affiliation: Beckman Coulter Incorporated, Miami, FL 33196, USA. vincent.shankey@coulter.com
authors.forename: T Vincent
authors.initials: TV
authors.lastname: Shankey
authors.forename: Meryl
authors.initials: M
authors.lastname: Forman
authors.forename: Paul
authors.initials: P
authors.lastname: Scibelli
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Cobb
authors.forename: Cecilia M
authors.initials: CM
authors.lastname: Smith
authors.forename: Rhonda
authors.initials: R
authors.lastname: Mills
authors.forename: Karen
authors.initials: K
authors.lastname: Holdaway
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Bernal-Hoyos
authors.forename: Mafalda
authors.initials: M
authors.lastname: Van Der Heiden
authors.forename: Jan
authors.initials: J
authors.lastname: Popma
authors.forename: Mike
authors.initials: M
authors.lastname: Keeney
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001402:B-Lymphocytes / Q000737:chemistry* / Q000276:immunology; D014408:Biomarkers, Tumor / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D002463:Cell Membrane Permeability; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007694:Killer Cells, Natural / Q000737:chemistry* / Q000276:immunology; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000276:immunology; D015203:Reproducibility of Results; D013194:Staining and Labeling; D013601:T-Lymphocytes / Q000737:chemistry* / Q000276:immunology; D016707:Tissue Fixation / Q000379:methods*; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human