title: Improved ZAP-70 assay using two clones, multiple methods of analysis and clinical correlation.
abstract: BACKGROUND
  In a companion methodological study, we compared two anti-ZAP-70 clones (1E7.2 AF 488 and SBZAP PE) and four selected methods of analysis. Clinical correlations are required for validation.
  
  
  METHODS
  Multicolor flow-cytometric evaluation of ZAP-70, CD38, CD69, CD26, CD49d, and CD27 was tested in 45 untreated-CLL patients. Four methods of ZAP-70 expression analysis and a scoring system were designed. A correlation analysis between ZAP-70 score, immunoglobulin heavy chain variable (IGHV) mutational status, fluorescence in situ hybridization, and these biomarkers was undertaken.
  
  
  RESULTS
  There is a strong correlation between ZAP-70 expression and IGHV mutational status. The scoring system for a single reagent (P = 0.0006 or 0.0002) favors the use of multiple methods of analysis. The combined score was substantially equivalent (P = 0.0003). There was also a correlation with del 13q14 (P = 0.017) and trisomy12 (P = 0.011). A correlation for CD38 and ZAP-70 score was seen using both 1E7.2 AF488 and SBZAP PE when ≥20% or ≥7% cutoff was used. A positive correlation was seen for CD49d expression using both reagents. CD26 showed a correlation with ZAP-70 expression, but it was dependent upon the method of analysis. CD69 and CD27 showed no statistically significant correlation.
  
  
  CONCLUSIONS
  In our study population, ZAP-70 expression is the better predictor of the IGHV mutational status. The correlation analysis confirms that the use of four methods of analysis with a single reagent or both reagents is superior to the use of a single method of analysis. The routine use of CD38, CD49d, and CD26 will require standardization.
authors.affiliation: Division of Cellular, Tissue and Gene Therapies, Center for Biologics Evaluation and Research, Food & Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA.
authors.forename: Heba A
authors.initials: HA
authors.lastname: Degheidy
authors.forename: David J
authors.initials: DJ
authors.lastname: Venzon
authors.forename: Mohammed Z H
authors.initials: MZ
authors.lastname: Farooqui
authors.forename: Fatima
authors.initials: F
authors.lastname: Abbasi
authors.forename: Diane C
authors.initials: DC
authors.lastname: Arthur
authors.forename: Wyndham H
authors.initials: WH
authors.lastname: Wilson
authors.forename: Adrian
authors.initials: A
authors.lastname: Wiestner
authors.forename: M A
authors.initials: MA
authors.lastname: Stetler-Stevenson
authors.forename: Gerald E
authors.initials: GE
authors.lastname: Marti
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal / Q000097:blood; D000954:Antigens, Surface / Q000097:blood / Q000276:immunology; D014408:Biomarkers, Tumor / Q000097:blood; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000592:standards*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D017404:In Situ Hybridization, Fluorescence; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood / Q000175:diagnosis / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000097:blood / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D014408:Biomarkers, Tumor; D007143:Immunoglobulin Heavy Chains; D051746:ZAP-70 Protein-Tyrosine Kinase