UNIT 9.22 Optimized Whole-Blood Assay for Measurement of ZAP-70 Protein Expression
Published Online: 1 JAN 2007
Copyright © 2006 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Cytometry
How to Cite
Shankey, T. V., Forman, M. and Scibelli, P. 2007. Optimized Whole-Blood Assay for Measurement of ZAP-70 Protein Expression. Current Protocols in Cytometry. 39:9.22:9.22.1–9.22.13.
- Published Online: 1 JAN 2007
- Published Print: JAN 2007
Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of small lymphocytes commonly expressing cell surface markers (CD5 and CD19) that are consistent with a population of B lymphocytes. This unit describes a technique to measure ZAP-70 protein expression in whole-blood specimens from CLL samples. The protocols presented include an optimized fixation/permeabilization technique that allows labeling of cell surface markers and intracellular ZAP-70 protein with significantly improved signal-to-noise ratio, an optimized combination of antibodies-fluorophores to maximize ZAP-70 expression levels, standardized methodology for instrument setup, including compensation, to improve inter- and intra-laboratory reproducibility, and a method to index ZAP-70 protein expression levels to internal positive and negative cell populations. Residual normal T and B cells function as internal positive and negative controls. These are used to index ZAP-70 protein expression levels in the CLL population.
- chronic lymphocytic leukemia;
- B lymphocytes;
- whole-blood assay;
- immunoglobulin heavy chain V-region (IgVH)