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 Zenontrade mark 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.


CONCLUSIONS
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.
Bojarska-Junak
Agnieszka
A
Department of Clinical Immunology, Medical University of Lublin, Lublin, Poland. agnieszkajunak@poczta.onet.pl
Giannopoulos
Krzysztof
K
Kowal
Malgorzata
M
Dmoszyńska
Anna
A
Roliński
Jacek
J
D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D014408:Biomarkers, Tumor / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D002463:Cell Membrane Permeability; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods*; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000096:biosynthesis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human