ZAP-70 by flow cytometry: a comparison of different antibodies, anticoagulants, and methods of analysis

O. G. Best, R. E. Ibbotson, A. E. Parker, Z. A. Davis, J. A. Orchard, D. G. Oscier

Research output: Contribution to journalArticlepeer-review

30 Citations (Scopus)

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. CONCLUSION: 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.
Original languageEnglish
Pages (from-to)235-241
Number of pages7
JournalCytometry. Part B, Clinical cytometry
Volume70B
Issue number4
DOIs
Publication statusPublished - 15 Jul 2006
Externally publishedYes

Keywords

  • Antibodies/*immunology Anticoagulants/*pharmacology Antigen-Antibody Reactions Biomarkers, Tumor/analysis/chemistry/immunology Disease Progression Flow Cytometry/*methods Humans Immunoglobulin Heavy Chains/genetics Immunoglobulin Variable Region/genetics Leukemia, Lymphocytic, Chronic, B-Cell/*diagnosis/genetics/metabolism Middle Aged Mutation Reproducibility of Results Sensitivity and Specificity Staining and Labeling Time Factors ZAP-70 Protein-Tyrosine Kinase/*analysis/drug effects/immunology

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