alexa Improved ZAP-70 assay using two clones, multiple methods of analysis and clinical correlation.


Journal of Blood Disorders & Transfusion

Author(s): Degheidy HA, Venzon DJ, Farooqui MZ, Abbasi F, Arthur DC, , Degheidy HA, Venzon DJ, Farooqui MZ, Abbasi F, Arthur DC,

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Abstract INTRODUCTION: In a companion methodological study, we compared two anti-ZAP-70 clones (1E7.2 AF 488 and SBZAP PE) and four selected methods of analysis. Clinical correlations are required for validation. METHODS: Multicolor flow-cytometric evaluation of ZAP-70, CD38, CD69, CD26, CD49d, and CD27 was tested in 45 untreated-CLL patients. Four methods of ZAP-70 expression analysis and a scoring system were designed. A correlation analysis between ZAP-70 score, immunoglobulin heavy chain variable (IGHV) mutational status, fluorescence in situ hybridization, and these biomarkers was undertaken. RESULTS: There is a strong correlation between ZAP-70 expression and IGHV mutational status. The scoring system for a single reagent (P = 0.0006 or 0.0002) favors the use of multiple methods of analysis. The combined score was substantially equivalent (P = 0.0003). There was also a correlation with del 13q14 (P = 0.017) and trisomy12 (P = 0.011). A correlation for CD38 and ZAP-70 score was seen using both 1E7.2 AF488 and SBZAP PE when ≥20\% or ≥7\% cutoff was used. A positive correlation was seen for CD49d expression using both reagents. CD26 showed a correlation with ZAP-70 expression, but it was dependent upon the method of analysis. CD69 and CD27 showed no statistically significant correlation. CONCLUSION: In our study population, ZAP-70 expression is the better predictor of the IGHV mutational status. The correlation analysis confirms that the use of four methods of analysis with a single reagent or both reagents is superior to the use of a single method of analysis. The routine use of CD38, CD49d, and CD26 will require standardization. Published 2011 Wiley-Liss, Inc.
This article was published in Cytometry B Clin Cytom and referenced in Journal of Blood Disorders & Transfusion

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