title: A robust ratio metric method for analysis of Zap-70 expression in chronic lymphocytic leukemia (CLL).
abstract: Since Zap-70 expression in chronic lymphocytic leukemia (CLL) cells correlates with a lack of somatic mutation of the immunoglobulin variable heavy chain (IgVH) genes, it has been proposed as a surrogate marker for disease prognosis. However, published studies of Zap-70 expression have used different commercial antibodies and analytic strategies. This study was undertaken to determine if any strategy was broadly applicable in a clinical flow cytometry laboratory. Expression of Zap-70 was determined in 37 CLL patients using four different commercial antibodies. T, NK, and CLL cells were identified by immunophenotyping along with Zap-70 expression. Data was analyzed in terms of both percent of CLL cells expressing Zap-70 and the ratio of Zap-70 expression in CLL cells compared to that in T + NK cells. Three Zap-70 antibodies showed wide ranges of Zap-70 expression as a percentage of tumor cells, while a fourth gave consistently elevated results. Comparing the percent Zap-70 expression with any two antibodies gave poor correlations (r(2) = 0.45-0.63). Our results indicated that the previous analytical strategies were not reproducible. A ratio metric is proposed, which gave better correlations (r(2) as high as 0.95) and would allow separation of CLL patients with elevated or decreased Zap-70 expression.
authors.affiliation: Department of Pathology/Laboratory Medicine, Oregon Health & Science University, Portland, OR, USA. bakkea@ohsu.edu
authors.forename: Antony C
authors.initials: AC
authors.lastname: Bakke
authors.forename: Zoe
authors.initials: Z
authors.lastname: Purtzer
authors.forename: Jose
authors.initials: J
authors.lastname: Leis
authors.forename: James
authors.initials: J
authors.lastname: Huang
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D000937:Antigen-Antibody Reactions; D014408:Biomarkers, Tumor / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D002138:Calibration; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000378:metabolism; D015203:Reproducibility of Results; 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