Optimized Whole‐Blood Assay for Measurement of ZAP‐70 Protein Expression

T. Vincent Shankey1, Meryl Forman1, Paul Scibelli1

1 Beckman Coulter, Inc., Miami, Florida
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.22
DOI:  10.1002/0471142956.cy0922s39
Online Posting Date:  January, 2007
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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)

     
 
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Table of Contents

  • Basic Protocol 1: Whole‐Blood Assay for Measurement of ZAP‐70 Protein Expression Using a Single‐Laser Flow Cytometer
  • Alternate Protocol 1: Whole‐Blood Assay for Measurement of ZAP‐70 Protein Expression Using a Dual‐Laser Flow Cytometer
  • Alternate Protocol 2: Three‐Antibody/Three‐Fluorophore Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Whole‐Blood Assay for Measurement of ZAP‐70 Protein Expression Using a Single‐Laser Flow Cytometer

  Materials
  • Compensation reagents:
    • CD45‐FITC/CD45‐PE, QuickCOMP 2 kit (Beckman‐Coulter, no. 177018)
    • CD45‐PECy5 (Beckman‐Coulter, no. IM265
    • CD45‐PECy7 (Beckman‐Coulter, no. IM3548)
  • Cell surface receptor antibody mix (CD5‐FITC, CD19‐PECy5, CD3+56‐PECy7)
  • Whole blood from a normal donor (Verify tube) and from CLL patients (sample), collected in EDTA, heparin, or ACD anticoagulant
  • 8% formaldehyde fixation solution (see recipe)
  • Triton X‐100 lyse/permeabilization buffer (see recipe), 37°C
  • Wash/diluent buffer (see recipe)
  • Analysis buffer: 0.1% (w/v) paraformaldehyde in PBS ( appendix 2A), pH 7.2
  • ZAP‐70‐PE (clone SBZAP; Beckman‐Coulter, no. 772587)
  • Fluorospheres, (e.g., Flow‐Check, Beckman‐Coulter, no. 6605359)
  • Fluorosphere microbeads (e.g., Flow‐Set, Beckman‐Coulter, no. 6607007)
  • PC7 (770) set up kit (Beckman‐Coulter, no. 6607121)
  • 12 × 75–mm polypropylene tubes

Alternate Protocol 1: Whole‐Blood Assay for Measurement of ZAP‐70 Protein Expression Using a Dual‐Laser Flow Cytometer

  • For cell surface antibody mix, CD19‐APC (substituted for CD19‐PECy5)
  • For compensation, CD45‐APC (substituted for CD45‐PECy5)
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Figures

Videos

Literature Cited

Literature Cited
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