ZAP‐70 Staining in Chronic Lymphocytic Leukemia

Neus Villamor1

1 Hospital Clínic de Barcelona, Barcelona, Spain
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.19
DOI:  10.1002/0471142956.cy0619s32
Online Posting Date:  May, 2005
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Abstract

Chronic lymphocytic leukemia (CLL) is the most common chronic leukemia in Western countries. The disease has an extremely variable clinical course, and several prognostic features have been identified to assess individual risk. The configuration of the immunoglobulin variable heavy‐chain gene (IgVH) is a strong predictor of the outcome. CLL patients with unmutated IgVH status have an aggressive clinical course and a short survival. Unfortunately, analysis of IgVH gene configuration is not available in most clinical laboratories. A small number of genes are differentially expressed between unmutated IgVH and mutated IgVH clinical forms of CLL. One of these genes is ZAP‐70, which is detected in leukemic cells from patients with the unmutated IgVH form of CLL. Flow cytometry presents advantages over other methods to detect ZAP‐70, and its quantification by flow cytometry has proved its predictive value. This unit focuses on protocols to quantify ZAP‐70 by flow cytometry in CLL.

Keywords: ZAP‐70; chronic lymphocytic leukemia; prognosis; B lymphocytes; mutational status of immunoglobulin variable heavy‐chain gene

     
 
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Table of Contents

  • Basic Protocol 1: Indirect Staining of Intracellular ZAP‐70
  • Alternate Protocol 1: Direct Immunofluorescence Staining of ZAP‐70
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Indirect Staining of Intracellular ZAP‐70

  Materials
  • Whole peripheral blood sample, mononuclear cells, or thawed cryopreserved cells
  • Fixation and Permeabilization Kit formulated to study intracellular antigens in leukemia cells (Caltag, Dako)
  • Unconjugated MAb against ZAP‐70 (e.g., clone 2F3.2, available from Upstate Biotechnology, Novus Biological, LabVision, Diagnostic Biosystems, or Acris Antibodies GmbH; or clone 1E7.2, available from Caltag and Upstate Biotechnology), appropriately titered (unit 4.2)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fluorescein isothiocyanate (FITC)‐conjugated polyclonal goat anti‐mouse IgG F(ab′) 2, appropriately titered
  • Normal mouse serum
  • Fluorochrome‐conjugated MAbs against B lymphocytes (CD19‐PerCP‐Cy5.5), T&NK cells (PE‐conjugated, e.g., CD3 and CD56), and CLL cells (CD5‐APC), appropriately titered
  • 12 × 75–mm polystyrene tubes
  • Flow cytometer with a 488‐nm HeNe laser and a 635‐nm red diode laser

Alternate Protocol 1: Direct Immunofluorescence Staining of ZAP‐70

  • Fluorochrome‐conjugated MAb against ZAP‐70 conjugated to Alexa Fluor 488 (Caltag), isotypic MAb conjugated to Alexa Fluor 488, MAb against B lymphocytes (CD19‐PerCP‐Cy5.5), MAb against T&NK cells (PE‐conjugated, e.g., CD3 and CD56), and MAb against CLL cells (CD5‐APC), appropriately titered
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Figures

Videos

Literature Cited

Literature Cited
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   Chen, L., Widhopf, G., Huynh, L., Rassenti, L., Rai, K.R., Weiss, A., and Kipps, T.J. 2002. Expression of ZAP‐70 is associated with increased B‐cell receptor signaling in chronic lymphocytic leukemia. Blood 100:4609‐4614.
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