title: Discordant results of flow cytometric ZAP-70 expression status in B-CLL samples if different gating strategies are applied.
abstract: BACKGROUND
  Recent studies have identified ZAP-70 expression status as an excellent prognostic parameter in chronic lymphocytic leukemia (CLL). ZAP-70 expression can be determined by direct antibody staining followed by flow cytometric analysis. However, there are currently several different gating strategies for analysis. We compared those strategies for ZAP-70 expression analysis.
  
  
  METHODS
  One hundred and one patients with B-CLL were analyzed employing a directly labeled alexa-fluor-488-ZAP-70-antibody. In 27 cases, we additionally measured and analyzed ZAP-70 expression, together with healthy controls as described previously.
  
  
  RESULTS
  Applying either T-/NK-cell isotype or healthy control analysis strategies on patient samples that were processed in parallel revealed discrepant results in 48% (12/25) of all cases. Taken together with the 74 B-CLL patients, who were analyzed with regard to average reference values, disconcordant results were obtained in 58% of the samples. We demonstrate that high variances in ZAP-70 T-/NK-cell staining within B-CLL patients, paired with a close proximity of ZAP-70 B-cell values to the suggested cut-off levels, may lead to interpretation difficulties of ZAP-70 status.
  
  
  CONCLUSIONS
  We conclude that different gating strategies for determining flow cytometric ZAP-70 expression status produce highly discordant results. Further standardization is required before ZAP-70 can be used as a reliable prognostic parameter in immunophenotyping of B-CLL.
authors.affiliation: Department of Hematology, Oncology and Immunology, Philipps-University of Marburg, Marburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Wilhelm
authors.forename: Andreas
authors.initials: A
authors.lastname: Neubauer
authors.forename: Cornelia
authors.initials: C
authors.lastname: Brendel
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D014408:Biomarkers, Tumor / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping; D053208:Kaplan-Meier Estimate; D007694:Killer Cells, Natural / Q000276:immunology; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000188:drug therapy / Q000276:immunology; D008875:Middle Aged; D012016:Reference Values; D015203:Reproducibility of Results; D015996:Survival Rate; D013601:T-Lymphocytes / Q000276:immunology; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology
chemical_list: D014408:Biomarkers, Tumor; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human