Improved ZAP‐70 assay using two clones, multiple methods of analysis and clinical correlation

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.