Unit

UNIT 9.22 Optimized Whole-Blood Assay for Measurement of ZAP-70 Protein Expression

  1. T. Vincent Shankey,
  2. Meryl Forman,
  3. Paul Scibelli

Published Online: 1 JAN 2007

DOI: 10.1002/0471142956.cy0922s39

Current Protocols in Cytometry

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.

Author Information

  1. Beckman Coulter, Inc., Miami, Florida

Publication History

  1. Published Online: 1 JAN 2007
  2. Published Print: JAN 2007

Abstract

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.

Keywords:

  • chronic lymphocytic leukemia;
  • ZAP-70;
  • B lymphocytes;
  • whole-blood assay;
  • immunoglobulin heavy chain V-region (IgVH)