Comparison of methods for determining zeta-chain associated protein-70 (ZAP-70) expression in patients with B-cell chronic lymphocytic leukemia (B-CLL)

Background: Zeta-chain associated protein of 70kDa (ZAP-70) is the most promising surrogate marker for the immunoglobulin heavy chain variable region (IgV(H)) mutation status in B-cell chronic lymphocytic leukemia (B-CLL). Crespo et al. proposed the detection of ZAP-70 by flow cytometry. Recently several novel monoclonal antibodies appeared on market. Methods: We compared different staining methods utilizing monoclonal antibodies (moAb) against ZAP-70: anti-ZAP-70 PE, clone 1E7.2, anti-ZAP-70 PE, clone 17A/P-ZAP70 directly stained with flourochrome as well as anti-ZAP-70 antibody, clone 2F3.2 stained with Zenon (TM) Alexa Fluor (R) 488 Labeling Kit. Additionally different reagents for permeabilization such as IntraPrep, FIX & PERM, Perm/Wash and 70% ethanol/paraformaldehyde were used to find the most clinically relevant and easy assay to determine ZAP-70 expression in B-CLL. Moreover we compared results of ZAP-70 expression assessed by whole blood protocol with those obtained using-peripheral mononuclear cells isolated from whole blood. Results: Anti-ZAP-70 moAb clone 17A/P-ZAP70 gave elevated. results for all B-CLL patients as well as healthy controls. Staining with anti-ZAP-70 moAb clone 1E7.2 and anti-ZAP-70 moAb clone 2F3.2 was effective in distinguishing negative and positive cells for ZAP-70 protein expression. Not statistically significant discrepancies of ZAP-70 expression were noticed between different fixation and permeabilization methods. Conclusion: Basing on results obtained during this study we can recommend use of anti-ZAP-70 PE, clone 1E7.2 moAb utilizing whole blood protocol with commercial kits for permeabilization as an easy method that brings compatible results to the original method proposed by Crespo et al. (c) 2006 International Society for Analytical Cytology.