Comparative Analysis of Flow Cytometric Techniques in Assessment of ZAP-70 Expression in Relation toIgVHMutational Status in Chronic Lymphocytic Leukemia

We compared 1 subjective and 5 objective flow cytometric methods to evaluate zeta-associated protein (ZAP-70) expression in relation to immunoglobulin heavy-chain variable-region (IgVH) gene mutational status in 154 samples from 125 patients with chronic lymphocytic leukemia (CLL). ZAP-70 expression determined by all methods used correlated with IgVH gene mutational status, but none of them demonstrated high concordance rates. Of the objective methods, ZAP-70 staining determined as a ratio of molecules of equivalent soluble fluorochrome intensity in CLL cells to that in normal B cells (ZAP-70+ staining in IgVH germline cases, 59%; ZAP-70- in IgVH mutated cases, 75%) or T cells (ZAP-70+ in IgVH germline cases, 66%; ZAP-70- in IgVH mutated cases, 57%) provides the best combination for assigning ZAP-70+ status to IgVH germline and ZAP-70- status to IgVH mutated cases. The subjective method based on ZAP-70 expression in natural killer/T cells gave a similar result, but reproducibility between laboratories may be difficult. Further studies on ZAP-70 expression in relation to clinical parameters may address whether ZAP-70 is an independent prognostic marker for CLL.