title: ZAP-70 by flow cytometry: a comparison of different antibodies, anticoagulants, and methods of analysis.
abstract: BACKGROUND
  The clinical course of chronic lymphocytic leukaemia (CLL) is variable. ZAP-70 expression is believed to provide prognostic information. The flow cytometric detection of ZAP-70 is difficult because it is an intracellular antigen with weak expression in CLL. Consensus has not been reached as to the best method for measurement.
  
  
  METHODS
  We analyzed 72 CLL patient samples for ZAP-70 expression and IgVH mutational status. Sensitivity and specificity of ZAP-70 expression against IgVH mutational status were assessed for two clones (2F3.2 and 1E7.2) and for four methods of analysis: percentage positivity (PP), comparing test to isotype control, ratio of geometric means of test and isotype control, and percentage and ratiometric methods comparing test and T/NK cell populations. The effects of anticoagulant, collection times, and time to analysis were also evaluated.
  
  
  RESULTS
  Sensitivity and specificity were 85 and 88%, respectively, for Upstate PP; 70 and 88% for Caltag PP; 89 and 91% for Upstate ratio; 89 and 88% for Caltag ratio. Intraobserver variability was smaller when ZAP-70 expression was assessed using a ratiometric approach rather than the percentage method. By 48 h, we observed an average decrease of 13% in the Caltag ratio in the heparin preserved samples compared to an increase of 3% in those collected in EDTA. Within the first 24-h period, a greater percent variability was observed in those samples collected into EDTA compared with heparin.
  
  
  CONCLUSIONS
  Our data support a rapid method for ZAP-70 measurement using commercially available fixation/permeabilization reagents, a conjugated antibody, and a ratiometric method of analysis that minimizes subjective interpretation of the results. This is a method of ZAP-70 assessment that could be included in a routine diagnostic CLL panel; however, the choice of anticoagulant and time of analysis after collection are critical factors in accurate assessment of ZAP-70 expression.
authors.forename: O G
authors.initials: OG
authors.lastname: Best
authors.forename: R E
authors.initials: RE
authors.lastname: Ibbotson
authors.forename: A E
authors.initials: AE
authors.lastname: Parker
authors.forename: Z A
authors.initials: ZA
authors.lastname: Davis
authors.forename: J A
authors.initials: JA
authors.lastname: Orchard
authors.forename: D G
authors.initials: DG
authors.lastname: Oscier
mesh_terms: D000906:Antibodies / Q000276:immunology*; D000925:Anticoagulants / Q000494:pharmacology*; D000937:Antigen-Antibody Reactions; D014408:Biomarkers, Tumor / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D018450:Disease Progression; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D009154:Mutation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D013997:Time Factors; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000187:drug effects / Q000276:immunology
chemical_list: D000906:Antibodies; D000925:Anticoagulants; D014408:Biomarkers, Tumor; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human