title: Feasibility of an easily applicable method of ZAP-70 measurement in chronic lymphocytic leukemia in the routine flow cytometry setting: A methodological approach.
abstract: Zeta-associated protein 70 (ZAP-70), determined by flow cytometry, has been advocated a surrogate marker of immunoglobulin (Ig)V(H) unmutated status in B chronic lymphocytic leukemia (CLL). The aim of the current study was to test the applicability of an easy flow cytometry protocol for ZAP-70 measurement in CLL samples. Samples from 61 CLL patients and 44 normal subjects were analyzed using a commercial ZAP-70 monoclonal antibody (1E7.2 clone) conjugated with phycoerythrin (PE) and Alexa 488 fluorochromes. Modifications of the published methods led to the structure of a simplified in-house method of ZAP-70 measurement. A three-color approach was used with CD19, CD3 gating comparing with the isotype control provided by the same manufacturer. The cutoff levels for ZAP-70 positivity were defined from a receiver operator characteristic curve in relation to the IgV(H) mutational status and from the ln normalized mean value +2 SD of normal controls. Using the 20% cutoff value for ZAP-70 positivity in CLL patients defined by the literature, ZAP-PE had a sensitivity of 55% and a specificity of 98% in predicting the IgV(H) mutational status, whereas the corresponding values for ZAP-Alexa were 55% and 84%, respectively. Using the 7% cutoff value for CD38 positivity, the sensitivity was 55%, whereas the specificity was 76%. ZAP-70-positive patients showed a shorter time to disease progression in comparison with ZAP-70-negative patients (p < 0.001). In conclusion, the 100% specific prediction of mutational status is accompanied by reduced sensitivity, thus limiting ZAP-70's applicability either as a single marker or combined with CD38 for the assessment of the mutational status of CLL.
authors.affiliation: Immunology Department and National Histocompatibility Centre, G Gennimatas General Hospital, Athens, Greece.
authors.forename: Freda
authors.initials: F
authors.lastname: Passam
authors.forename: Varvara
authors.initials: V
authors.lastname: Tachynopoulou
authors.forename: Dimitra
authors.initials: D
authors.lastname: Skoumi
authors.forename: Aliki
authors.initials: A
authors.lastname: Tsompanakou
authors.forename: Aikaterini
authors.initials: A
authors.lastname: Stavropoulos-Giokas
authors.forename: Chrysanthi
authors.initials: C
authors.lastname: Vadikolia
authors.forename: Achilles
authors.initials: A
authors.lastname: Anagnostopoulos
authors.forename: Georgios
authors.initials: G
authors.lastname: Paterakis
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000032:analysis; D000328:Adult; D000368:Aged; D018941:Antigens, CD19; D015415:Biomarkers / Q000032:analysis; D017252:CD3 Complex; D016022:Case-Control Studies; D018450:Disease Progression; D005240:Feasibility Studies; D005260:Female; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000201:enzymology / Q000401:mortality; D008214:Lymphocytes; D008297:Male; D008722:Methods; D008875:Middle Aged; D011379:Prognosis; D012372:ROC Curve; D012015:Reference Standards; D016019:Survival Analysis; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis*
chemical_list: D018941:Antigens, CD19; D015415:Biomarkers; D017252:CD3 Complex; D051746:ZAP-70 Protein-Tyrosine Kinase; D051997:ADP-ribosyl Cyclase 1