ZAP-70 by flow cytometry: a comparison of different antibodies, anticoagulants, and methods of analysis
| Autor: | Rachel E. Ibbotson, Jenny A. Orchard, Zadie Davis, David Oscier, A. E. Parker, Oliver Giles Best |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Oncology
medicine.medical_specialty Histology Time Factors medicine.drug_class Immunoglobulin Variable Region Biology Sensitivity and Specificity Antibodies Pathology and Forensic Medicine Flow cytometry Antigen-Antibody Reactions Antigen Internal medicine medicine Biomarkers Tumor Humans ZAP-70 Protein-Tyrosine Kinase medicine.diagnostic_test Staining and Labeling Anticoagulant Anticoagulants Reproducibility of Results Cell Biology Heparin Middle Aged Flow Cytometry Isotype Leukemia Lymphocytic Chronic B-Cell Immunology Mutation biology.protein Disease Progression Immunoglobulin heavy chain Antibody Geometric mean Immunoglobulin Heavy Chains medicine.drug |
| Zdroj: | Cytometry. Part B, Clinical cytometry. 70(4) |
| ISSN: | 1552-4949 |
| Popis: | 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. q 2006 International Society |
| Databáze: | OpenAIRE |
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