Comparative analysis of flow cytometric techniques in assessment of ZAP-70 expression in relation to IgVH mutational status in chronic lymphocytic leukemia

We compared I subjective and 5 objective flow cytometric methods to evaluate zeta-associated protein (ZAP-70) expression in relation to immunoglobulin heavy-chain variable-region (IgV(H)) gene mutational status in 154 samples from 125 patients with chronic lymphocytic leukemia (CLL). ZAP-70 expression determined by all methods used correlated with IgV(H) 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 IgV(H) germline cases, 59%; Z4P-70- in IgV(H) mutated cases, 75%) or T cells (Z4P-70+ in IgV(H) germline cases, 66%; ZAP-70- in IgV(H) mutated cases, 57%) provides the best combination for assigning ZAP-70+ status to IgV(H) germline and ZAP-70- status to IgV(H) mutated 9 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 Z4P-70 expression in relation to clinical parameters may address whether Z4P-70 is an independent prognostic marker for CLL.