Measurement of ZAP-70 Expression in CLL Using an Optimized Flow Cytometric Assay for ZAP-70 Protein Levels in Whole Blood Samples.

ZAP-70 protein plays a critical role in the initiation of T-cell signaling through the TCR, and also regulates B cell differentiation. Studies aimed at understanding the relationship of maturation to disease course in the B-cell form of Chronic Lymphocytic Leukemia (B-CLL) have demonstrated a correlation of disease progression with IgV_H configuration (germ line vs mutated). Flow cytometry-based assays for ZAP-70 expression in CLL patient samples have been previously reported, and generally show poor separation of ZAP-70 levels in samples considered as positive from negative, making interpretation of these data difficult, and reducing the likely agreement between laboratories. Using a unique fixation and permeabilization technique in conjunction with an optimal ZAP-70-PE antibody conjugate, we have developed a whole blood assay for the measurement of ZAP-70 protein expression which results in a significantly increased S/N of 18–24 (normal blood T-cells to B-cells) compared to previously reported flow cytometric assays (S/N of 3 to 8). Based on these data, we have developed a 5 antibody/4 color whole blood assay (CD5-FITC, ZAP-70-PE, CD19-PE-Cy5, CD3+CD56-PECy7) which includes surface labeling before fixation and ZAP-70 labeling after permeabilization. Critical assay details including pre and post staining stability, instrument standardization, gating strategies and data interpretation will be presented and discussed. Using this optimized assay, we have undertaken a multi-institutional study to determine the inter-laboratory variability of ZAP-70 measurements using normal donors, and an assessment of the ability of this assay to increase the separation of B-CLL ZAP-70 positive from negative populations. Finally, we have developed a method to index differential ZAP-70 expression using internal controls, which obviates the use of quadrant analysis techniques.